Novel 3&#39; end caps, 5&#39; end caps and combinations thereof for therapeutic rna

ABSTRACT

The disclosure relates to nucleic acids that contain modifications at the 5′-end, 3′-end or 5′-end and 3′-ends, and compounds that can be used to make the modified nucleic acids are disclosed. The modified nucleic acids have improved expression, lower immunogenicity and improved stability compared to unmodified nucleic acids.

This application is a divisional of U.S. application Ser. No. 16/802,014filed 26 Feb. 2020, which is a divisional of U.S. application Ser. No.15/536,516 filed 15 Jun. 2017, which is a U.S. National Phase filing ofInternational Application No. PCT/M2015/059697 filed 16 Dec. 2015, whichclaims priority to U.S. Application No. 62/092,627 filed 16 Dec. 2014,the contents of which are incorporated herein by reference in theirentirety.

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Aug. 12, 2022, isnamed PAT056073-US-DIV02_SL_ST26.xml and is 24,576 bytes in size.

BACKGROUND

Isolated nucleic acids, particularly those that encode proteins, areattractive candidates for a variety of clinical applications. Inparticular RNAs that encode proteins, such as mRNAs, provide a number ofpotential advantages for clinical applications. For example, RNA can betransfected into cell in vivo, in vitro or ex vivo to induce expressionof desired therapeutic or diagnostic proteins for treating or diagnosingdisease. However, the potential of RNA therapeutics is limited bystability and half-life of the RNA as well as by the level of expressionof the encoded protein.

Naturally occurring mRNAs contain a 5′ cap structure which helpsstabilize the RNA and is fundamental to eukaryotic gene expression(Shuman, S. et al Mol. Microbiol. 1995, 17, 405-410). These mRNAs alsocontain a 3′ poly A tail. Both modification stabilize and improvetranslation of the encoded protein. The 5′-cap structure found at the 5′end of eukaryotic messenger RNAs (mRNAs) and many viral RNAs consists ofa N⁷-methylguanosine nucleoside linked to the 5′-terminal nucleoside ofthe pre-mRNA via a 5′-5′ triphosphate linkage (Shatkin, A. J. Cell 1976,2, 645-53; Shuman, S. Prog. Nucleic Acid Res. Mol. Biol. 2001, 66, 1-40;Decroly, E. et al PLoS Pathog. 2011, 7, e1002059). This cap structurefulfills many roles that ultimately lead to mRNA translation. RNAcapping is also important for other processes, such as RNA splicing andexport from the nucleus and to avoid recognition of mRNA by the cellularinnate immunity machinery (Daffis, S. et al Nature 2010, 468, 452-6;Züst, R. et al Nat. Immunol. 2011, 12, 137-43). A number of syntheticcap analogs have been described. See, e.g., WO2009/149253,WO2011/015347.

Synthetic mRNAs are typically prepared by enzymatic synthesis using RNApolymerase and a DNA template followed by enzymatic addition of the5′-cap and the 3′-end (Peyrane, F. et al Nucleic Acids Res. 2007, 35,e26). However, the process is expensive and difficult to control andtherefore undesirable for commercial scale production.

Thus, a need exists for RNAs that are modified at the 5′-end, the 3′-endor the 5′-end and 3′-end that can be efficiently produced and that haveimproved expression of products (e.g., protein) encoded by the RNA,lower immunogenicity and/or improve stability.

SUMMARY OF THE INVENTION

This disclosure relates to nucleic acid molecules (e.g., RNA and DNAmolecules) that contain modifications at the 5′-end, the 3′-end, or boththe 5′-end and the 3-end. The nucleic acid molecule is preferably an RNAmolecule that encodes a product, such as a polypeptide or nucleic acid,and more preferably is an mRNA including mRNAs encoding Cas9 proteinsfor CRISPR genome editing technologies and/or the 3′-end of an sgRNA (orcrRNA and tracrRNA) for CRISPR genome editing technologies.

In one aspect, this disclosure relates to a compounds of Formula I andsalts (preferably a pharmaceutically acceptable salt) thereof:

wherein

Z₁ is

a cap 0, a cap 1,

with the proviso that when Z₁ is

cap 0 or cap 1, -A₁-R⁵ and -A₃-R⁸ are not both —OH, or A₂ is not null;

Z₂ is null or a linking moiety, selected from the group consisting of—O—, —S—, optionally substituted lower alkyl, optionally substitutedaminoalkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted cycloalkyl, optionally substitutedcycloalkenyl, optionally substituted heterocyclyl,

A₁ and A₃ are independently selected from the group consisting of null,NH, S, and O;

A₂ is null or selected from the group consisting of >CR⁶R⁷, >NR⁶,>NNR⁶R⁷, >NOR⁶, >S, and >O;

Y is null or a linking moiety selected from the group consisting ofoptionally substituted lower alkyl, optionally substituted alkenyl,optionally substituted alkynyl, —(CH₂)_(n)OR¹⁵, —(CH₂)_(n)COOR¹⁵, and—(CH₂)_(n)C(O)NR¹²;

R¹ is selected from the group consisting of H, optionally substitutedcycloalkyl, optionally substituted cycloalkenyl, optionally substitutedaryl, optionally substituted heteroaryl, and optionally substitutedheterocyclyl;

R² is selected from the group consisting of H, —OH, optionallysubstituted alkyl, —C(O)NR¹²R¹³ and —NR¹²R¹³;

R⁵, R⁶, and R⁸ are independently selected from the group consisting ofH, optionally substituted alkyl, polyamine, PEGs, —(CH₂)_(n1)NR¹²R¹³,—(CH₂)_(n1)NR¹⁴C(O)R¹⁵, —(CH₂)_(n1)OR¹⁵, —(CH₂)_(n1)C(O)OR¹⁵,—(CH₂)_(n1)C(O)R¹⁵, —(CH₂)_(n1)C(O)NR¹²R¹³,—O—(CH₂)_(n3)—C(O)—(NR¹²)₂—C(O)—X₂,—O—(CH₂)_(n3)—C(O)—[NR¹²—C(O)—(CH₂)_(n3)]₁₋₃—X₂, or R⁶ and R⁸ togetherform a ring that is optionally substituted and contains 10-80 ring atomsin which 10-40 ring atoms can be hetero atoms, or R⁶ and R⁷ togetherform a 3-8 membered ring that is optionally substituted and in which 1to 6 ring atoms can be hetero atoms;

R⁷, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently selected from thegroup consisting of H, optionally substituted lower alkyl, andoptionally substituted acyl;

R⁹ is selected from the group consisting of H and optionally substitutedlower alkyl;

R¹⁰ is selected from the group consisting of H, —NR¹²R¹³, and —OR¹⁶;

n is 1 to 4;

n1 is zero to 10;

n2 is 1 to 12;

n3 is 1 to 8;

X is selected from the group consisting of O, S, NH, and optionallysubstituted alkanediyl;

X₁ is selected form the group consisting of

R³ and R⁴ are independently selected from the group consisting of H and—OR¹⁶, or R³ and R⁴ together form O-Q-O;

Q is selected from the group consisting of —CH₂— and —C(Me)₂-;

X₂ is selected from the group consisting of affinity moiety anddetection moiety, and

X_(n) is a nucleobase.

In some aspect, the compound of Formula I is modified at both the 5′-endand 3′-end and is a compound of Formulas II, III, IV, or a salt(preferably a pharmaceutically acceptable salt) thereof,

The variables in Formulas II, III and IV are as defined in Formula I,with the proviso that -A₁-R⁵ and -A₃-R⁸ are not both —OH, or A₂ is notnull. In some embodiments, the compound is of Formula II, III or IV,with the proviso that that -A₁-R⁵ and -A₃-R⁸ are not both —OH, or A₂ isnot null.

In other aspects, the compounds of Formula I is modified at the 5′-endand unmodified at 3′-end, and is a compound of Formula VI, Formula VII,Formula VIII, or a salt (preferably a pharmaceutically acceptable salt)thereof,

The variables in Formulas VI, VII and VIII are as defined in Formula I,with the proviso that -A₁-R⁵ and -A₃-R⁸ are both —OH, and A₂ is null.

In certain preferred embodiments the compound is of any of Formulas I-IVand VI-VIII, wherein Y is an alkyl linking moiety, preferably a loweralkyl linking moiety such as (—CH₂—); and R¹ is a substituted aryl orsubstituted heteroaryl, for example aryl substituted with phenyl orheteroaryl substituted with phenyl. In particularly preferredembodiments, —Y—R¹ can be

In other aspects, the compounds of Formula I is modified at the 3′-endand unmodified at 5′-end, and is a compound of Formula V or a salt(preferably a pharmaceutically acceptable salt) thereof

In Formula V, *Z₁ is selected from a group consisting of

a cap 0 and a cap 1, and the other variables are as described in FormulaI, with the proviso that A₂ is not null, or -A₁-R⁵ and -A₃-R⁸ are notboth —OH.

The compounds of Formulas I-VIII contain a nucleobase, X_(n), which canbe any desired nucleobase, such as adenine, guanine, cytosine, uracil ora modified nucleobase such as pseudouracil. A preferred nucleobase isadenine.

The RNA in compounds of Formulas I-VIII can be any desired RNA molecule,but preferable encodes a product, such as a protein.

In other aspects, the disclosure relates to guanosine or purinederivatives that can be used to prepare 5′-end modified nucleic acids(e.g., RNAs) disclosed herein. In particular aspects, the disclosurerelates to a compound of Formula IX, X, XI or salt (pharmaceuticallyacceptable salt) thereof:

In each of Formulas IX, X and XI,

Z is selected from a group consisting of —OH,

n4 is 0-2; and the other variables are as defined in Formula I. Incertain preferred embodiments of this aspect, Y is an alkyl linkingmoiety, preferably a lower alkyl linking moiety such as (—CH₂—); and R¹is a substituted aryl or substituted heteroaryl, for example arylsubstituted with phenyl or heteroaryl substituted with phenyl. Inparticularly preferred embodiments —Y—R¹ can be

The invention also relates to methods of using and making the compoundsdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph and corresponding capped mRNAs depictingluciferase activity of enzymatically capped HPLC purified mRNA (Cap-1)compared to HPLC purified chemically capped mRNAs 1, 2, and 3.

FIG. 2 is a bar graph and corresponding capped mRNAs depictingluciferase activity of enzymatically capped HPLC purified mRNA (Cap-0)compared to HPLC purified chemically capped mRNAs 1, 2, and 3.

FIG. 3 is a bar graph and corresponding capped mRNAs depicting leptinexpression data with chemically capped HPLC Purified mRNAs Cap-1 andCap-2.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art.

This disclosure relates to nucleic acid molecules (e.g., RNA and DNAmolecules) that contain modifications at the 5′-end, the 3′-end, or boththe 5′-end and the 3-end. The nucleic acid molecule is preferably an RNAmolecule that encodes a product, such as a polypeptide or nucleic acid,and more preferably is an mRNA. The RNA or mRNA that is modified asdisclosed herein can be produced using any desired method, suchenzymatic or chemical synthesis. The 5′-end modification comprise capstructures based on guanosine or purine that increase expression ofproducts (e.g., protein) encoded by the RNA, lower immunogenicity and/orimprove stability of the RNA. The 3′-end modifications comprisemodification of the cis-diol at the 3′ and 2′ positions of the terminalribose of the RNA, for example by inserting a ring atom between thesepositions or replacing one or both hydroxyl groups with othersubstituents, to increase expression of products (e.g., protein) encodedby the RNA and/or improve stability of the RNA (e.g., by slowing therate of degradation). If desired, the 3′-end modification can include avariety of functional moieties, such as an affinity moiety or adetection moiety. Compounds that contain such moieties are particularlyuseful for, for example, imaging, biochemical analysis andbioconjugation.

As described and exemplified herein, RNAs have been prepared thatcontain the 5′-end cap structures based on guanosine or purine that aredisclosed herein. These modified RNAs were shown to bind to eIF4E and tobe translated in cells. 3′-end RNAs were also prepared and had increasedhalf-life and expression in comparison to unmodified RNA.

In one aspect, this disclosure relates to RNA molecules that contain a5′-end cap structure, a 3′-end modification, or a 5′-end cap structureand a 3′-end modification. Such RNA molecules are compounds of Formula Iand salts (preferably pharmaceutically acceptable salts) thereof:

wherein

-   -   Z₁ is

a cap 0, a cap 1,

with the proviso that when Z₁ is

cap 0 or cap 1, -A₁-R⁵ and -A₃-R⁸ are not both —OH, or A₂ is not null;

Z₂ is null or a linking moiety, selected from the group consisting of—O—, —S—, optionally substituted lower alkyl, optionally substitutedaminoalkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted cycloalkyl, optionally substitutedcycloalkenyl, optionally substituted heterocyclyl,

A₁ and A₃ are independently selected from the group consisting of null,NH, S, and O;

A₂ is null or selected from the group consisting of >CR⁶R⁷, >NR⁶,>NNR⁶R⁷, >NOR⁶, >S, and >O;

Y is null or a linking moiety selected from the group consisting ofoptionally substituted lower alkyl, optionally substituted alkenyl,optionally substituted alkynyl, —(CH₂)_(n)OR¹⁵, —(CH₂)_(n)COOR¹⁵, and—(CH₂)_(n)C(O)NR¹²;

R¹ is selected from the group consisting of H, optionally substitutedcycloalkyl, optionally substituted cycloalkenyl, optionally substitutedaryl, optionally substituted heteroaryl, and optionally substitutedheterocyclyl;

R² is selected from the group consisting of H, —OH, optionallysubstituted alkyl, —C(O)NR¹²R¹³ and —NR¹²R¹³;

R⁵, R⁶, and R⁸ are independently selected from the group consisting ofH, optionally substituted alkyl, polyamine, PEGs, —(CH₂)_(n1)NR¹²R¹³,—(CH₂)_(n1)NR¹⁴C(O)R¹⁵, —(CH₂)_(n1)OR¹⁵, —(CH₂)_(n1)C(O)OR¹⁵,—(CH₂)_(n1)C(O)R¹⁵, —(CH₂)_(n1)C(O)NR¹²R¹³,—O—(CH₂)_(n3)—C(O)—(NR¹²)₂—C(O)—X₂,—O—(CH₂)_(n3)—C(O)—[NR¹²—C(O)—(CH₂)_(n3)]₁₋₃—X₂, or R⁶ and R⁸ togetherform a ring that is optionally substituted and contains 10-80 ring atomsin which 10-40 ring atoms can be hetero atoms, or R⁶ and R⁷ togetherform a 3-8 membered ring that is optionally substituted and in which 1to 6 ring atoms can be hetero atoms;

R⁷, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently selected from thegroup consisting of H, optionally substituted lower alkyl, andoptionally substituted acyl;

R⁹ is selected from the group consisting of H and optionally substitutedlower alkyl;

R¹⁰ is selected from the group consisting of H, —NR¹²R¹³, and —OR¹⁶;

n is 1 to 4;

n1 is zero to 10;

n2 is 1 to 12;

n3 is 1 to 8;

X is selected from the group consisting of O, S, NH, and optionallysubstituted alkanediyl;

X₁ is selected form the group consisting of

R³ and R⁴ are independently selected from the group consisting of H and—OR¹⁶, or R³ and R⁴ together form O-Q-O;

Q is selected from the group consisting of —CH₂— and —C(Me)₂-;

X₂ is selected from the group consisting of affinity moiety anddetection moiety, and

X_(n) is a nucleobase.

In certain preferred embodiments the compound is of Formula I, wherein

Z₁ is

Y is an alkyl linking moiety, preferably a lower alkyl linking moietysuch as (—CH₂—); and

R¹ is a substituted aryl or substituted heteroaryl, for example arylsubstituted with phenyl or heteroaryl substituted with phenyl. Inparticularly preferred embodiments —Y—R¹ can be

In other preferred embodiments the compound is of Formula I, wherein-A₁-R⁵ and -A₃-R⁸ are not both —OH, or A₂ is not null. In anotherpreferred embodiment, -A₁-R⁵ and -A₃-R⁸ are not both —OH, and A₂ is notnull.

In some aspect, the compound of Formula I is modified at both the 5′-endand 3′-end and is a compound of Formulas II, III or IV, or a saltthereof,

The variables in Formulas II, III and IV are as defined in Formula I,with the proviso that -A₁-R⁵ and -A₃-R⁸ are not both —OH, or A₂ is notnull. In some embodiments, the compound is of Formula II, III or IV,with the proviso that that -A₁-R⁵ and -A₃-R⁸ are not both —OH, or A₂ isnot null.

In certain preferred embodiments of this aspect, Y is an alkyl linkingmoiety, preferably a lower alkyl linking moiety such as (—CH₂—); and R¹is a substituted aryl or substituted heteroaryl, for example arylsubstituted with phenyl or heteroaryl substituted with phenyl. Inparticularly preferred embodiments, —Y—R¹ can be

In other aspects, the compounds of Formula I is modified at the 5′-endand unmodified at 3′-end, and is a compound of Formula VI, Formula VII,Formula VIII, or a salt thereof,

The variables in Formulas VI, VII and VIII are as defined in Formula I,with the proviso that -A₁-R⁵ and -A₃-R⁸ are both —OH, and A₂ is null. Incertain preferred embodiments of this aspect, Y is an alkyl linkingmoiety, preferably a lower alkyl linking moiety such as (—CH₂—); and R¹is a substituted aryl or substituted heteroaryl, for example arylsubstituted with phenyl or heteroaryl substituted with phenyl. Inparticularly preferred embodiments, —Y—R¹ can be

In other aspects, the compounds of Formula I is modified at the 3′-endand unmodified at 5′-end, and is a compound of Formula V or a saltthereof

In Formula V, *Z₁ is selected from a group consisting of

a cap 0 and a cap 1, and the other variables are as described in FormulaI, with the proviso that A₂ is not null, or -A₁-R⁵ and -A₃-R⁸ are notboth —OH.

The compounds of Formulas I-VIII contain a nucleobase, X_(n), which canbe any desired nucleobase, such as adenine, guanine, cytosine, uracil ora modified nucleobase such as pseudouracil. A preferred nucleobase isadenine.

The RNA in compounds of Formulas I-VIII can be any desired RNA molecule,but preferable encodes a product, such as a protein. Suitable proteinsinclude therapeutic proteins, such as antibodies or antigen-bindingfragments thereof, receptors, cytokines and growth factors; antigens,such as those from pathogens or tumor cells. The RNA, e.g., mRNA, maycontain a ribose-phosphate backbone with the common nucleobases adenine,cytosine, guanine and uracil bonded to the 1″ position of the ribosering. If desired, one or more modified bases may be included in the RNAto the desired degree. For example, between 0.1% and 100% of thenucleobases can be a modified base, such as pseudouracil. If desired,the RNA molecule can contain one or more phosphoramidate,phosphorothioate, methylphosphonate, phosphoroselenoate or othersuitable linkages.

In other aspects, the disclosure relates to guanosine or purinederivatives that can be used to prepare 5′-end modified nucleic acids(e.g., RNAs) disclosed herein.

In particular aspects, the disclosure relates to a compound of FormulaIX, X, XI or salt (pharmaceutically acceptable salt) thereof:

In each of Formulas IX, X and XI,

Z is selected from a group consisting of —OH,

n4 is 0-2; and the other variables are as defined in Formula I. Incertain preferred embodiments of this aspect, Y is an alkyl linkingmoiety, preferably a lower alkyl linking moiety such as (—CH₂—); and R¹is a substituted aryl or substituted heteroaryl, for example arylsubstituted with phenyl or heteroaryl substituted with phenyl. Inparticularly preferred embodiments, —Y—R¹ can be

In preferred compounds of Formula IX, R² is preferably optionallysubstituted amino; X is preferably O; and/or X₁ is preferably

wherein R³ and R⁴ are as defined in Formula I.

Method of Using End Capped Nucleic Acids

The end capped nucleic acids disclosed herein can be used for a varietyof purposes, including to induce the production of a desired protein bya cell for diagnostic or therapeutic purposes. For example, an endcapped mRNA that encodes a protein can be transfected into a cell invivo or in vitro. Suitable methods for transfection of mRNAs are wellknown in the art and include, for example, methods that use cationicpolymers, calcium phosphate or cationic lipids to facilitatetransfection, as well as direct injection, biolistic particle delivery,electroporation, laser irradiation, sonoporation and magneticnanoparticle methods. See, e.g., Kim et al, Anal Bioanal Chem.397:3173-3178 (2010).

The cell to be transfected is preferably an animal cell, such as from amammal, a fish, a bird and more preferably a human. Suitable animalsubjects include, for example, cattle, pigs, horses, deer, sheep, goats,bison, rabbits, cats, dogs, chickens, ducks, turkeys, and the like.

End capped RNA molecules of the invention can also be delivered to cellsex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy), followed byre-implantation of the cells into a patient, usually after selection forcells which have been transfected with the end capped RNA molecule. Theappropriate amount of cells to deliver to a patient will vary withpatient conditions, and desired effect, which can be determined by askilled artisan. Preferably, the cells used are autologous, i.e., cellsobtained from the patient being treated.

The end capped nucleic acids (e.g., mRNAs) can be used in the genomeediting technology known as CRISPRs (clustered regularly interspacedshort palindromic repeats) which are part of bacterial anti-viraldefence systems and which can be used to induce targeted genome editingin eukaryotic cells. When used as a tool for genome editing the type IICRISPR system is typically composed of 2 or 3 components: the Cas9protein which is an RNA dependent DNA endonuclease and either one or 2RNAs per DNA strand cleavage site (the natural 2 RNA system includes aCRISPR-RNA (crRNA) and a trans-activating RNA (tracrRNA) while in thesingle RNA system the 2 crRNA and tracrRNA have been re-engineered intoa single functional transcript called a single guide RNA (sgRNA)). Astandard system for CRISPR editing can encode the sgRNA or multiple RNAsand the Cas9 protein in a lentiviral vector. However a challenge withviral vectors is that there is a limit to the size of additional genesthat can be encoded by them and the large Cas 9 proteins whose sizeranges from ˜1000-2000 amino acids in length can be difficult to encodein viral vectors. An all RNA CRISPR system using an in vitro transcribedCas9 mRNA co-formulated with an in vitro transcribed sgRNA (or crRNA+tracrRNA) could be co-formulated for transfection into cells invivo, in vitro or ex vivo in order to specifically edit the genomes ofthe transfected cells. The end capped mRNAs disclosed herein are wellsuited for this application.

End capped nucleic acids can also be used for a variety of biochemicaland analytical purposes. For example, the end capped nucleic acids canbe used for imaging studies, to study the metabolism of RNA and to studythe interaction of RNA with other biomolecules.

Synthetic Schemes

The compounds of the present invention may be prepared by the routesdescribed in the following Schemes or Examples.

Scheme 1: Chemically capped mRNA of the general structure I, as definedin Formula VI can be obtained by reacting mRNA-5′-monophosphate withimidazole activated cap structures of the general structure II in asuitably buffered aqueous saline solution (buffers: HEPES, TRIS, IVIES,PBS) at a suitable pH, ranging from 5.5-7.5, in presence or absence oforganic solvents, such as DMF and DMSO, in the presence of a suitableLewis-acidic activator such as MnCl₂, NiCl₂, ZnCl₂.

Scheme 2: Compounds of the general structure II, where Y and R¹ aredefined as in Formula I, can be prepared from by treatment of phosphatesIII (tributylammonium salts) with imidazole or N-methyl-imidazole, asuitable tertiary phosphine, such as triphenylphosphine, a suitableoxidant, such as 2,2′-dipyridyldissulfide, and a suitable base, such astriethylamine in a suitable solvent, such as DMF, DMSO, NMP,trimethylphosphate at a suitable temperature, such as room temperature(a) M. Lewdorowicz, Y. Yoffe, J. Zuberek, J. Jemielity, J. Stepinski, R.Kierzek, R. Stolarski, M. Shapira, E. Darzynkiewicz, RNA 2004, 10, 1469;b) R. Worch, J. Stepinski, A. Niedzwiecka, M. Jankowska-Anyszka, C.Mazza, S. Cusack, R. Stolarski, E. Darzynkiewicz, NucleosidesNucleotides and Nucleic Acids 2005, 24, 1131; c) M. Warminski, J.Kowalska, J. Buck, J. Zuberek, M. Lukaszewicz, C. Nicola, A. N. Kuhn, U.Sahin, E. Darzynkiewicz, J. Jemielity, Bioorg. Med. Chem., 2013, 23,3753).

Scheme 3: Compounds of the general structure III, where Y and R¹ aredefined as in Formula I, can be prepared by reaction of activatedphosphates IV with triethylammonium phosphate, or a similar phosphatesalt, in a suitable solvent such as DMF, DMSO, or water in the presenceof a lewis acid such as zinc chloride, magnesium chloride or manganesechloride at a suitable temperature such as room temperature (a) M.Lewdorowicz, Y. Yoffe, J. Zuberek, J. Jemielity, J. Stepinski, R.Kierzek, R. Stolarski, M. Shapira, E. Darzynkiewicz, RNA 2004, 10, 1469;b) R. Worch, J. Stepinski, A. Niedzwiecka, M. Jankowska-Anyszka, C.Mazza, S. Cusack, R. Stolarski, E. Darzynkiewicz, NucleosidesNucleotides and Nucleic Acids 2005, 24, 1131; c) M. Warminski, J.Kowalska, J. Buck, J. Zuberek, M. Lukaszewicz, C. Nicola, A. N. Kuhn, U.Sahin, E. Darzynkiewicz, J. Jemielity, Bioorg. Med. Chem., 2013, 23,3753).

Compounds of the general structure III, where Y and R¹ are defined as inFormula I, can be prepared by alkylation of V using a suitablealkylation reagent, such as an alkyl halide, triflate, or mesylate in asuitable solvent such as DMF, DMSO, or NMP at a suitable reactiontemperature ranging from room temperature to 60° C.

When X═O, compounds of the general structure III, where Y and R¹ aredefined as in Formula I, can also be prepared by reaction of VI withdiphosphoric acid tetrachloride in a suitable solvent such astrimethylphosphate at a suitable temperature ranging from 0° C. to roomtemperature (J. Emsley, J. Moore, P. B. Udy, J. Chem. Soc. (A) Inorg.Phys. Theor. 1971, 2863).

When X═NH, compounds of the general structure III, where Y and R¹ aredefined as in Formula I, can also be prepared by reaction of IV withimido-bis(phosphoryldichloride) in a suitable solvent such astrimethylphosphate at a suitable temperature ranging from 0° C. to roomtemperature (A. M. Rydzik, M. Kulis, M. Lukaszewicz, J. Kowalska, J.Zuberek, Z. M. Darzynkiewicz, E. Darzynkiewicz, J. Jemielity, Bioorganic& Med. Chem. 2012, 20, 1699).

When X═CH₂, compounds of the general structure III, where Y and R¹ aredefined as in Formula I, can also be prepared by reaction of IV withmethylenebis(phosphonic dichloride) in a suitable solvent such astrimethylphosphate at a suitable temperature such as 0° C. or roomtemperature (a) M. Honcharenko, M. Zytek, B. Bestas, P. Moreno, J.Jemielity, E. Darzynkiewicz, C. I. E. Smith, R. Stroemberg, Bioorg. Med.Chem., 2013, 21, 7921; b) M. Kalek, J. Jemielity, Z. M. Darzynkiewicz,E. Bojarska, J. Stepinski, R. Stolarski, R. E. Davis, E. Darzynkiewic,Bioorg. Med. Chem. 2006, 14, 3223; c) M. Kalek, J. Jemielity, J.Stepinski, R. Stolarski, E. Darzynkiewics, Tetrahedron Lett. 2005, 46,2417).

Scheme 4: Compounds of the general structure IV, where Y and R¹ aredefined as in Formula I, can be prepared from by treatment of phosphatesVII (tributylammonium salts) with imidazole, a suitable tertiaryphosphine, such as triphenylphosphine, a suitable oxidant, such as2,2′-dipyridyldissulfide, and a suitable base, such as triethylamine ina suitable solvent, such as DMF or DMSO at a suitable temperature, suchas room temperature (P. C. Joshi, M. F. Aldersley, D. V. Zagorevskii, J.P. Ferris, Nucleosides, Nucleotides and Nucleic Acids 2012, 31, 7, 536).

Scheme 5: Compounds of the general structure VII, where Y and R¹ aredefined as in Formula I, can be prepared by alkylation of VIII using asuitable alkylation reagent, such as an alkyl halide, triflate, ormesylate in a suitable solvent such as DMF or DMSO at a suitablereaction temperature ranging from room temperature to 50° C.

Compounds of the general structure VIII, where Y and R¹ are defined asin Formula I, can be obtained by reaction of X with a suitablephophorylating agent such as phosphoryl chloride in a suitable solventsuch as trimethylphosphate at a suitable temperature ranging from 0° C.or room temperature.

Compounds of the general structure VII, where Y and R¹ are defined as inFormula I, can also be prepared by reaction of IX with a suitablephophorylating agent such as phosphoryl chloride in a suitable solventsuch as trimethylphosphate at a suitable temperature ranging from 0° C.to room temperature.

Compounds of the general structure IX, where Y and R¹ are defined as inFormula I, can be prepared by alkylation of X using a suitablealkylation reagent, such as an alkyl halide, triflate, or mesylate in asuitable solvent such as DMF or DMSO at a suitable reaction temperatureranging from room temperature to 50° C.

Scheme 6: Compounds of the general structure VI, where Y and R¹ aredefined as in Formula I, can be prepared by alkylation of X using asuitable alkylation reagent, such as an alkyl halide, triflate, ormesylate in a suitable solvent such as DMF or DMSO at a suitablereaction temperature ranging from room temperature to 50° C.

Scheme 7: When X═Br, Cl, I and A=CH₃, compounds of the general structureXI and XII can be prepared from XIII and XIV, respectively, by radicalhalogenation using a suitable radical initiator such asazodiisobutyronitrile and a halide donor such as N-bromosuccinimide,N-Bromoacetamide, N-chlorosuccinimide, N-iodosuccinimide, bromide,chloride, or iodide, in a suitable solvent such as carbon tetrachlorideat a suitable reaction temperature such at 85° C. (a) K. Ziegler, A.Spath, E. Schaaf, W. Schumann, E. Winkelmann, Ann. 1942, 551, 80; b) A.Nechvatal, Advances in Free-Radical Chemistry (London) 1972, 4, 175).

When X═Br, Cl, I and A=CH₂OH, compounds of the general structure XI andXII can be prepared from XIII and XIV, respectively, by reaction with asuitable phosphine such as triphenylphosphine and a suitable oxidantsuch as carbon tetrabromide, carbon tetrachloride, N-iodosuccinimide ina suitable solvent such as dichloromethane at a suitable reactiontemperature such as room temperature (a) R. Appel, Angew. Chem. Int. Ed.1979, 14, 801; b) Cadogan, J, ed. (1979). Organophosphorus Reagents inOrganic Synthesis. London: Academic Press).

When X═Br, Cl, I and A=CH₂OH, compounds of the general structure XI andXII, can be prepared from XIII and XIV, respectively, by treatment witha suitable acid such as HBr (48% aq.), HCl (12 M aq.), or HI (aq.) (a)M. Uchida, F. Tabusa, M. Komatsu, S. Morita, T. Kanbe, K. Nakagawa,Chem. Pharm. Bull. 1985, 33, 3775; b) V. Boekelheide, G. K. Vick, J. Am.Chem. Soc. 1956, 78, 653; c) K. M. Doxsee, M. Feigel, K. D. Stewart, J.W. Canary, C. B. Knobler, D. J. Cram, J. Am. Chem. Soc. 1987, 109,3098.).

When X═OMs, OTf and A=CH₂OH, compounds of the general structure XI andXII can be prepared from XIII and XIV, respectively, by reaction with asuitable reagent such as MsCl, (Ms)₂O, (Tf)₂O in the presence of asuitable base such as a tertiary amine, e.g. triethylamine, in asuitable solvent such as dichloromethane.

When A=CH₂OH, compounds of the general structure XIII can be preparedfrom the corresponding acid (A=COOH) by reaction with a suitablereductant such as borane dimethylsulfide adduct or lithium aluminumhydride in a suitable solvent such as tetrahydrofurane at at suitablereaction temperature such as room temperature (a) N. G. Gaylord,Reduction with Complex Metal Hydrides, Wiley, N.Y., 1956, 322; b) H. C.Brown, W. Korytnyk, J. Am. Chem. Soc. 1960, 82, 3866).

When A=CH₃, CH₂OH compounds of the general structures XIII and XIV canbe prepared by Suzuki crosscoupling reaction using the suitable arylsubstrates XV-XVII as starting material (N. Miyaura, A. Suzuki, Chem.Rev. 1995, 95, 2457).

If B═Br, I, OTf, the cross coupling reaction involves reaction of XV orXVI with boron reagents XVII (C═B(OH)₂, Bpin) using a suitable catalystsuch as Sphos palladacycle G2(Chloro(2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II))and a suitable base such as K₂CO₃ in a suitable solvent mixture such asDMF/H₂O at a suitable temperature such as room temperature, 50° C., or80° C. (T. E. Barder, S. D. Walker, J. R. Martinelli, S. L. Buchwald, S.L. J. Am. Chem. Soc. 2005, 127 4685; b) R. A. Altman, S. L. Buchwald,Nature Protocols 2007, 2, 3115-3121).

If B═B(OH)₂, Bpin, the cross coupling reaction involves reaction of XVor XVI with reagents XVII (C═Br,I,OTf) using a suitable catalyst such asSphos palladacycle G2(Chloro(2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II))and a suitable base such as K₂CO₃ in a suitable solvent mixture such asDMF/H₂O at a suitable temperature such as room temperature, 50° C., or80° C. Compounds XVI-XVII are commercially available.

Scheme 8: Chemically 3′-modified mRNA of the general structure XVIII, asdefined in Formula V, can be obtained by reacting RNA with the generalstructure XX with NaIO₄ in a suitably buffered aqueous solution(buffers: NaOAc, TRIS, PBS, MES, HEPES), at a suitable pH, ranging from5.0-7.5, at a temperature ranging from 0° C. to room temperature,followed by treatment with suitable nucleophiles such as hydrazines,acylhydrazones, hydroxylamines, 1,2-aminothiols, amines.

Chemically 3′-modified mRNA of the general structure XVIII, as definedin Formula V can be obtained by reacting RNA with the general structureXX with NaIO₄ in a suitably buffered aqueous solution (buffers: NaOAc,TRIS, PBS, MES, HEPES), at a suitable pH, ranging from 5.0-7.5, at atemperature ranging from 0° C. to room temperature, followed bytreatment with a suitable amine nucleophile such as hydrazines,acylhydrazones, hydroxylamines, amines followed by treatment with asuitable reducing agent, such as NaCNBH₃, at temperatures ranging fromroom temperature to 37° C.

RNA of the general structure XIX, as defined in Formula I can beobtained by reacting RNA with the general structure XX with NaIO₄ in asuitably buffered aqueous solution (buffers: NaOAc, TRIS, PBS, MES,HEPES), at a suitable pH, ranging from 5.0-7.5, at a temperature rangingfrom 0° C. to room temperature, followed by treatment with suitablenucleophile such Meldrum's acid.

Scheme 9: 5′3′-bismodified RNA of the general structures XXI and XXII,as defined below, can be obtained from 3′-modified RNA of the structureXXIII and XXIV, respectively, by reaction with guanosine derivatives II,under conditions described in Scheme 1.

Scheme 10: Chemically capped mRNA of the general structure XXV can beobtained by reacting mRNA-5′-monophosphate with imidazole activated capstructures of the general structure XXVI in a suitably buffered aqueoussaline solution (buffers: HEPES, TRIS, MES, PBS) at a suitable pH,ranging from 5.5-7.5, in presence or absence of organic solvents, suchas DMF and DMSO, in the presence of a suitable Lewis-acidic activatorsuch as MnCl₂, NiCl₂, ZnCl₂.

Exemplary 5′ cap structures that can be prepared include,

Scheme 11: Compounds of the general structure XXVI, where Y and R¹ aredefined as in Formula I, can be prepared from by treatment of phosphatesXXVII (tributylammonium salts) with imidazole or N-methyl-imidazole, asuitable tertiary phosphine, such as triphenylphosphine, a suitableoxidant, such as 2,2′-dipyridyldissulfide, and a suitable base, such astriethylamine in a suitable solvent, such as DMF, DMSO, NMP,trimethylphosphate at a suitable temperature, such as room temperature(a) M. Lewdorowicz, Y. Yoffe, J. Zuberek, J. Jemielity, J. Stepinski, R.Kierzek, R. Stolarski, M. Shapira, E. Darzynkiewicz, RNA 2004, 10, 1469;b) R. Worch, J. Stepinski, A. Niedzwiecka, M. Jankowska-Anyszka, C.Mazza, S. Cusack, R. Stolarski, E. Darzynkiewicz, NucleosidesNucleotides and Nucleic Acids 2005, 24, 1131; c) M. Warminski, J.Kowalska, J. Buck, J. Zuberek, M. Lukaszewicz, C. Nicola, A. N. Kuhn, U.Sahin, E. Darzynkiewicz, J. Jemielity, Bioorg. Med. Chem., 2013, 23,3753).

Scheme 12: Compounds of the general structure XXVII, where Y, R¹ and Z₂are defined as in Formula I, can be prepared by reaction of activatedphosphates XXIX with triethylammonium phosphate, or a similar phosphatesalt, in a suitable solvent such as DMF, DMSO, or water in the presenceof a lewis acid such as zinc chloride, magnesium chloride or manganesechloride at a suitable temperature such as room temperature (a) M.Lewdorowicz, Y. Yoffe, J. Zuberek, J. Jemielity, J. Stepinski, R.Kierzek, R. Stolarski, M. Shapira, E. Darzynkiewicz, RNA 2004, 10, 1469;b) R. Worch, J. Stepinski, A. Niedzwiecka, M. Jankowska-Anyszka, C.Mazza, S. Cusack, R. Stolarski, E. Darzynkiewicz, NucleosidesNucleotides and Nucleic Acids 2005, 24, 1131; c) M. Warminski, J.Kowalska, J. Buck, J. Zuberek, M. Lukaszewicz, C. Nicola, A. N. Kuhn, U.Sahin, E. Darzynkiewicz, J. Jemielity, Bioorg. Med. Chem., 2013, 23,3753).

When X═O, compounds of the general structure XXVII, where Y, R¹ and Z₂are defined as in Formula I, can also be prepared by reaction of XXVIIIwith diphosphoric acid tetrachloride in a suitable solvent such astrimethylphosphate at a suitable temperature ranging from 0° C. to roomtemperature (J. Emsley, J. Moore, P. B. Udy, J. Chem. Soc. (A) Inorg.Phys. Theor. 1971, 2863).

When X═NH, compounds of the general structure XXVII, where Y, R¹ and Z₂are defined as in Formula I, can also be prepared by reaction of XXVIIIwith imido-bis(phosphoryldichloride) in a suitable solvent such astrimethylphosphate at a suitable temperature ranging from 0° C. to roomtemperature (A. M. Rydzik, M. Kulis, M. Lukaszewicz, J. Kowalska, J.Zuberek, Z. M. Darzynkiewicz, E. Darzynkiewicz, J. Jemielity, Bioorganic& Med. Chem. 2012, 20, 1699).

When X═CH₂, compounds of the general structure XXVII, where Y, R¹ and Z₂are defined as in Formula I, can also be prepared by reaction of XXVIIIwith methylenebis(phosphonic dichloride) in a suitable solvent such astrimethylphosphate at a suitable temperature such as 0° C. or roomtemperature (a) M. Honcharenko, M. Zytek, B. Bestas, P. Moreno, J.Jemielity, E. Darzynkiewicz, C. I. E. Smith, R. Stroemberg, Bioorg. Med.Chem., 2013, 21, 7921; b) M. Kalek, J. Jemielity, Z. M. Darzynkiewicz,E. Bojarska, J. Stepinski, R. Stolarski, R. E. Davis, E. Darzynkiewic,Bioorg. Med. Chem. 2006, 14, 3223; c) M. Kalek, J. Jemielity, J.Stepinski, R. Stolarski, E. Darzynkiewics, Tetrahedron Lett. 2005, 46,2417).

Compounds of the general structure XXIX (Z=imidazole,N-methylimidazolium), where Y, R¹ and Z₂ are defined as in Formula I,can be prepared from by treatment of phosphates XXIX (Z═OH,tributylammonium salts) with imidazole or N-methyl-imidazole, a suitabletertiary phosphine, such as triphenylphosphine, a suitable oxidant, suchas 2,2′-dipyridyldissulfide, and a suitable base, such as triethylaminein a suitable solvent, such as DMF, DMSO, NMP, trimethylphosphate at asuitable temperature, such as room temperature (a) M. Lewdorowicz, Y.Yoffe, J. Zuberek, J. Jemielity, J. Stepinski, R. Kierzek, R. Stolarski,M. Shapira, E. Darzynkiewicz, RNA 2004, 10, 1469; b) R. Worch, J.Stepinski, A. Niedzwiecka, M. Jankowska-Anyszka, C. Mazza, S. Cusack, R.Stolarski, E. Darzynkiewicz, Nucleosides Nucleotides and Nucleic Acids2005, 24, 1131; c) M. Warminski, J. Kowalska, J. Buck, J. Zuberek, M.Lukaszewicz, C. Nicola, A. N. Kuhn, U. Sahin, E. Darzynkiewicz, J.Jemielity, Bioorg. Med. Chem., 2013, 23, 3753).

Compounds of the general structure XXIX (Z═OH), where Y, R¹ and Z₂ aredefined as in Formula I, can also be prepared by reaction of XXVIII witha suitable phophorylating agent such as phosphoryl chloride in asuitable solvent such as trimethylphosphate at a suitable temperatureranging from 0° C. to room temperature.

Compounds of the general structure XXVII, where Y, R¹ and Z₂ are definedas in embodiment Formula I, can also be prepared by crosscouplingreaction using the suitable aryl substrates and compound XXX as startingmaterial (N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457).

Compound XXX can be obtained by alkylation of the commercially available8-bromo-3-methyl-1H-purine-2,6(3H,7H)-dione (A=H) or8-bromo-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (A=Me) using a suitablealkylation reagent, such as an alkyl halide, triflate, or mesylate in asuitable solvent such as DMF, DMSO, or NMP at a suitable reactiontemperature ranging from room temperature to 60° C.

Scheme 13: Compounds of the general structure XXVIII, where Y, R¹ and Z₂are defined as in Formula I, can be obtained from amides of the generalstructure XXXII by cyclization using an appropriate base, such as sodiumtert-butoxide, potassium tert-butoxide, or sodium iso-propoxide, in asuitable solvent, such as ethanol, isopropanol, or THF, at temperaturesranging from 50-100° C.

Compounds of the general structure XXIX, where Y, R¹ and Z₂ are definedas in Formula I, can be obtained from mono- or dialkyl phosphanes andphosphates of the general structure XXXI by hydrolysis using a suitablelewis acid, such as trimethylsilyl bromide, boron tribromide, oraluminum trichloride, in a solvent such as DMF, DMSO, NMP, or THF, attemperatures ranging from 0° C. to 30° C.

Compounds of the general structure XXXI, where Y, R¹ and Z₂ are definedas in embodiment Formula I, can be obtained from amides of the generalstructure XXXII by cyclization using an appropriate base, such as sodiumtert-butoxide, potassium tert-butoxide, or sodium iso-propoxide, in asuitable solvent, such as ethanol, isopropanol, or THF, at temperaturesranging from 50-100° C.

Scheme 14: Compounds of the general structure XXXII, where Y, R¹ and Z₂are defined as in embodiment XXX, can be obtained by acylation of XXXIIIwith the appropriate carboxylic acid of the linker unit using a suitableactivator such as HATU, HBTU,O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, or(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate anda suitable base such as triethylamine or Hunig's base, in a suitablesolvent, such as DMF, DMSO, or NMP at temperatures ranging from roomtemperature to 50° C.

Compounds of the general structure XXXIII, where Y, R¹ is defined as inembodiment Formula I, can be obtained by reduction of XXXIV (where Y isthe corresponding carboxylic acid of the substituent R1) using areducing agent, such as lithium aluminum hydride or Red-Al, in asuitable solvent such as THF, diethylether, or dioxane at temperaturesranging from room temperature to 120° C.

Compounds of the general structure XXXIV, can be obtained by acylationof commercially available 5,6-diaminopyrimidine-4(3H)-one (XXXV) withthe appropriate carboxylic acid to introduce subsitutent R¹ using asuitable activator such as HATU, HBTU,O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, or(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate anda suitable base such as triethylamine or Hunig's base, in a suitablesolvent, such as DMF, DMSO, or NMP at temperatures ranging from roomtemperature to 50° C.

Scheme 15: 5′3′-bismodified RNA of the general structures XXXVI andXXXVII, as defined above, can be obtained from 3′-modified RNA of thestructure XL and XLI, respectively, by reaction with guanosinederivatives XXVI, under conditions described in Scheme 1. The synthesisof XXVI is described in Scheme 10.

Definitions

The term “acyl” refers to an optionally substituted alkyl carbonyl,optionally substituted arylcarbonyl. Examples of such acyl groupsinclude acetyl, benzoyl, and the like.

The term “affinity moiety” refers to a molecule that specifically bindsto a molecule of interest, such as a protein, or nucleic acid or othermolecule. Examples of affinity moiety include, but are not limited to,biotin and digoxigenin.

The term “alkanediyl” refers divalent radicals of the general formulaC_(n)H_(2n) derived from aliphatic hydrocarbons. Unless specifiedotherwise, such alkanediyls include substituted alkanediyls. Suitableexamples include methanediyl (—CH₂—), ethanediyl (—CH₂—CH₂—), and thelike.

The terms “alkenyl” and “alkynyl” as used herein, alone or incombination, refers to aliphatic straight-chain or branched hydrocarbonchains that contain 1 to 20 carbon atoms and include one or more unitsof unsaturation. Alkenyl contains at least one carbon-carbon doublebond, but no carbon-carbon triple bonds. Alkynyl contains at least onecarbon-carbon triple bond. Preferred alkenyl and alkynyl group maycomprise from 2 to 10 carbon atoms or from 2 to 6 carbon atoms. Suitablealkenyl groups include, for example, vinyl, allyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl,6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl,6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl,5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl,3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, or9-decenyl. Suitable alkynyl groups include, for example, ethynyl,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl,5-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 3-octynyl, 4-octynyl,5-octynyl, 6-octynyl, 7 -octynyl, 1-nonylyl, 2-nonynyl, 3-nonynyl,4-nonynyl, 5-nonynyl, 6-nonynyl, 7-nonynyl, 8-nonynyl, 1-decynyl,2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl,8-decynyl, or 9-decynyl. Alkenyl and Alkynyl groups may be optionallysubstituted as described herein.

The term “alkyl,” as used herein, alone or in combination, refers to analiphatic straight-chain or branched, saturated hydrocarbon chaincontaining from 1 to 20 carbon atoms. In certain embodiments, the alkylgroup may comprise from 1 to 10 carbon atoms. In further embodiments,the alkyl group may comprise from 1 to 6 carbon atoms. Alkyl groups maybe optionally substituted as described herein. Examples of alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl, noyl and thelike.

The term “amino” refers to the group —NH₂.

The term “aminoalkyl” refers to an alkyl substituted with a primary,secondary or tertiary amino group. Preferred aminoalkyl groups includethose groups having one or more primary, secondary and/or tertiary aminegroups, and from 1 to about 12 carbon atoms, more preferably 1 to about8 carbon atoms, still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms.Examples of such aminoalkyl include aminomethyl, aminoethyl, and thelike.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic hydrocarbon ring system containing at least onearomatic ring. The aromatic ring system can be a fused ring systemincluding aromatic or non-aromatic hydrocarbon rings or an aromatic ringsystem that include a non-aromatic hydrocarbon ring. The term “aryl”embraces aromatic groups such as phenyl, naphthyl, anthracenyl, andphenanthryl. Aryl may be optionally substituted as described herein.

The term “cap 0” refers to the caps in which the only methylation is inthe ^(m7)G. The term “cap 1” refers to caps with additional methylationin N₁. The generalized cap structure is represented as^(m7)G(5′)ppp(5′)N₁ ^(m)pN₂ ^(m)pN₃p . . . where N is any nucleotide,preferably a purine or a pyrimidine, p is a phosphate group and m is amethyl group. The ^(m7)G, containing a methyl group at the N⁷ positionof guanosine, is at the extreme 5′ terminus of the mRNA. Methylations atthe N₁ and N₂ positions, on the other hand, are substitutions at the2′-OH group of the ribose moiety. The various cap structures areclassified on the basis of the number of methyl groups they contain(Banerjee, A. K. Microbiological Rev., 1980, 44, 175).

The term “cycloalkenyl,” as used herein, alone or in combination withother terms, represents, unless otherwise stated, cyclic versions of“alkenyl” containing at least one carbon-carbon double bond, withpreferably 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, or 10 carbonatoms, preferably 3 to 6 carbon atoms, forming a ring. The term“cycloalkenyl” is also meant to include bicyclic versions thereof. Ifbicyclic rings are formed it is preferred that the respective rings areconnected to each other at two adjacent carbon atoms, however,alternatively the two rings are connected via the same carbon atom,i.e., they form a spiro ring system or they form bridged ring systems.Examples of cycloalkenyl include cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, 1,3-cyclohexenyl,cycloheptenyl, cycloheptatrienyl, cyclooctenyl, cyclononeyl,cyclodecenyl, bicyclo[2.2.1]-2-heptenyl, bicyclo[2.2.1]-2-octenyl, orbicyclo[4.4.0]-2-decenyl. A cycloalkenyl group may be optionallysubstituted as described herein.

The term “cycloalkyl,” as used herein, alone or in combination withother terms, represents, unless otherwise stated, cyclic versions of“alkyl” including bicyclic and polycyclic alkyl groups. The rings of abicyclic or polycyclic ring system can be fused are linked through asingle shared atom i.e., they form a spiro ring system or a bridged ringsystem. A cycloalkyl can preferably contain 3 to 10 carbon atoms, i.e.,3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, preferably 3 to 6 carbon atomsforming a ring. Examples of suitable cycloalkyl groups includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclononyl, or cylcodecyl. Examples of suitable cycloalkylgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, spiro[3,3]heptyl, spiro[3,4]octyl,spiro[4,3]octyl, bicyclo[4.1.0]heptyl, bicyclo[3.2.0]heptyl,bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl,bicyclo[4.2.0]octyl, and the like. A cycloalkyl group may be optionallysubstituted as described herein.

The term “detection moiety” refers to chemical moieties that can bereadily detected using suitable methods. Examples of detection moietyinclude, but are not limited to, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials.

The term “half-life” (T_(1/2)) relates to the period of time which isneeded to eliminate half of the activity, amount, or number ofmolecules. In the context of the present invention, the half-life of RNAis indicative of the stability of said RNA.

The term “hetero atom” refers to nitrogen, oxygen and sulfur.

The term “heteroaryl,” as used herein, is an aryl in which one or morecarbon ring atoms are independently replaced by O, S, and N. In certainembodiments, the heteroaryl may comprise from 5 to 7 carbon atoms. Theterm also embraces fused polycyclic groups wherein heterocyclic ringsare fused with aryl rings, wherein heteroaryl rings are fused with otherheteroaryl rings, wherein heteroaryl rings are fused withheterocycloalkyl rings, or wherein heteroaryl rings are fused withcycloalkyl rings. Examples of heteroaryl groups include pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl,indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl,quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl,benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl,benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl,tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl,thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplarytricyclic heterocyclic groups include carbazolyl, benzidolyl,phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyland the like. Heteroaryl may be optionally substituted as describedherein.

The term “heterocyclyl” means a cycloalkyl group as defined above inwhich from 1 to 3 carbon atoms in the ring are independently replaced byO, S, or N. Examples of such heterocyclyl groups are pyrrolidinyl,imidazolidinyl, oxazolidinyl, thiazolidinyl, tetrahydrofuranyl,piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydropyranyl, dioxanyl, indolinyl, isoindolinyl,dihydrobenzofuranyl, dihydro-isobenzofuranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, tetrahydroquinoxalinyl, chromanyl,isochromanyl, dihydrobenzooxazinyl, dihydrobenzothiazinyl, ordihydrobenzodioxinyl. A heterocyclyl may be optionally substituted asdescribed herein.

The term “immunogenicity” refers to the capacity to induce an immunereaction.

The term “linking group” refers to a divalent radical that links twoother moieties. The linking group generally will contain between 1 and40 atoms and can be, for example, an alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocycle linking group,each of which can optionally be substituted as described herein. Forexample, suitable linking groups include methanediyl (—CH₂—), ethanediyl(—CH₂—CH₂—), ethenediyl (—CH═CH—), ethynediyl (—C≡C—), —CH₂CH₂CH₂—,—CH₂CH(CH₃)—, —C(CH₃)₂—, —(C₆H₄)—, —(C₆H₁₀)—, and the like. Additionalexamples of suitable linking groups include but not limited to

The term “lower,” as used herein, alone or in a combination, where nototherwise specifically defined, means containing from 1 to and including6 carbon atoms.

The term “nucleobase” refers to nitrogen-containing bases that arecomponents of nucleotides. Nucleobases include, for example, the primarynucleobases cytosine, guanine, adenine, thymine, uracil; pseudouracil;as well as modified nucleobases, such as, m5C (5-methylcytidine), m5U(5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um(2′-0-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine);Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine);i6A (N6-isopentenyladenosine); ms2i6A(2-methylthio-N6isopentenyladenosine); io6A(N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m11 (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine);f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine);m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G (archaeosine); D (dihydrouridine);m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluricjine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm (2′-O-methylinosine); m4C(N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G(N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm (5-formyl-2′-O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-0-dimethyl adenosine)irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); or ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine,7-substituted derivatives thereof, dihydrouracil, pseudouracil,2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C₁-C₆)-alkyluracil,5-methyluracil, 5-(C₂-C₆)-alkenyluracil, 5-(C₂-C₆)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C₁-C₆)-alkylcytosine, 5-methylcytosine,5-(C₂-C₆)-alkenylcytosine, 5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine,5-fluorocytosine, 5-bromocytosine, N²-dimethylguanine, 7-deazaguanine,8-azaguanine, 7-deaza-7-(C2-C6)alkynylguanine, 8-hydroxyguanine,6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine,2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, and the like.

When a group is defined to be “null,” what is meant is that the group isabsent.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, halogen, hydroxy, amino, lower alkylamino,arylamino, amido, nitro, thiol, lower alkylthio, lower haloalkylthio,lower perhaloalkylthio, arylthio, sulfonate, sulfonic acid,trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H, nitrile, CF₃,cycloalkyl, pyridinyl, thiophene, furanyl, lower carbamate, halophenyl,hydroxyphenyl, haloalkyl and hydroxyalkyl. Two substituents may bejoined together to form a five-, six-, or seven-membered aromatic ornon-aromatic carbocyclic or heterocyclic ring containing one to threeheteroatoms, for example forming methylenedioxy or ethylenedioxy. Anoptionally substituted group may be unsubstituted (e.g., —CH₂CH₃), fullysubstituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F) orsubstituted at a level anywhere in-between fully substituted andmonosubstituted (e.g., —CH₂CF₃). Where substituents are recited withoutqualification as to substitution, both substituted and unsubstitutedforms are encompassed. Where a substituent is qualified as“substituted,” the substituted form is specifically intended.Additionally, different sets of optional substituents to a particularmoiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The compounds according to the present invention can be provided in“pharmaceutically acceptable preparations”. Such compositions maycontain salts, buffers, preserving agents, carriers and optionally othertherapeutic agents. “Pharmaceutically acceptable salts” comprise saltsthat are physiologically compatible and preferably non-toxic. Forexample, acid addition salts which may, for example, be formed by mixinga solution of compounds with a solution of a pharmaceutically acceptableacid such as hydrochloric acid, sulfuric acid, fumaric acid, maleicacid, succinic acid, acetic acid, benzoic acid, citric acid, tartaricacid, carbonic acid or phosphoric acid. Furthermore, where the compoundcarries an acidic moiety, suitable pharmaceutically acceptable saltsthereof may include alkali metal salts (e.g., sodium or potassiumsalts); alkaline earth metal salts (e.g., calcium or magnesium salts);and salts formed with suitable organic ligands (e.g., ammonium,quaternary ammonium and amine cations formed using counteranions such ashalide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkylsulfonate and aryl sulfonate). Illustrative examples of pharmaceuticallyacceptable salts include, but are not limited to, acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate,bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate,camphorate, camphor sulfonate, camsylate, carbonate, chloride, citrate,clavulanate, cyclopentanepropionate, digluconate, dihydrochloride,dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate,formate, fumarate. gluceptate, glucoheptonate, gluconate, glutamate,glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate,maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate,N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate),palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate,phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate,salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, undecanoate, valerate, and the like(see, for example, Berge, S. M. et al J. Pharm. Sci. 1977, 66, 1-19).

The term “PEG” refers to polymers having the repeat unit

where n2 is 2 to 12. Straight or branched polyethylene glycol polymersare encompassed by this term, and includes the monomethylether ofpolyethylene glycol (mPEG). The term “PEG” also encompasses Newkome-typedendritic molecules such as

and also

PEGs are commercially available in a number of formulations (e.g.,Carbowax™ (Dow Chemical, Midland, Mich.), Poly-G® (Arch Chemicals,Norwalk Conn.), and Solbase).

The term “polyamine” refers to amine compounds containing at least twoamino groups which have at least one amino hydrogen atom. Examples ofpolyamine include, but not limited to polyethylene imine,polypropylene-imine, poly-vinylamine, polyallylamine, ethylene diamine,hexamethylene diamine, diethylene triamine, triethylene tetramine,tetraethylene pentamine and bishexamethylene triamine.

In the Formulas disclosed herein, the representation

refers to an RNA in 5′→3′ orientation.

EXAMPLES

Materials and Methods: All reagents were purchased from commercialsources and used without treatment, unless otherwise indicated. NMRspectra were run on Bruker AVANCE 400 MHz or 500 MHz NMR spectrometersusing ICON-NMR, under TopSpin program control. ¹³C NMR and ³¹P NMRspectra were recorded. Spectra were measured at 298K, unless indicatedotherwise, and were referenced relative to the solvent resonance.Chemical shifts (δ) are reported in ppm, and signals are described as s(singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).Mass spectra were acquired on LC-MS systems using electrosprayionization methods from a range of instruments of the configurationsshown below. [M+H⁺]⁺ refers to protonated molecular ion of the chemicalspecies. [M−H⁺]⁻ refers to a deprotonated molecular ion of the chemicalspecies.

LCMS Method 1 RXNMON_ACIDC

Instrument Agilent 1100 HPLC; Waters Micromass ZQ Mass SpectrometerColumn Sunfire C18 3.5 μm 30 × 3.0 mm, 3.5 μm Column 40° C. TemperatureEluents A: H₂O + 0.05% TFA, B: acetonitrile Flow Rate 2 ml/min Gradient5%-95% B in 1.7 min

LCMS Method 2 RXNMON Acidic_Polar_=RXNMON Acidic_Polar_PosNeg

Instrument Agilent 1100 HPLC; Waters Micromass ZQ Mass SpectrometerColumn Sunfire C18 3.5 μm 30 × 3.0 mm, 3.5 μm Column 40° C. TemperatureEluents A: H₂O + 0.05% TFA, B: acetonitrile Flow Rate 2 ml/min Gradient1% to 30% B in 1.20 min, 30% to 95% B in 0.65 min

LCMS Method 4 RXNMON_Basic_Polar

Instrument Agilent 1100 HPLC; Waters Micromass ZQ Mass SpectrometerColumn Xbridge C18 3.5 μm 30 × 3.0 mm, 3.5 μm Column 40° C. TemperatureEluents A: H₂O + 5 mM ammonium hydroxide, B: acetonitrile Flow Rate 2ml/min Gradient 1% to 30% B in 1.20 min, 30% to 95% B in 0.65 min

LCMS Method 5 SQ4mRNAcap_FIA Acidic

Instrument Waters Acquity UPLC with SQ detector Column Acquity BEH C181.7 μm 2.1 × 50 mm Column 50° C. Temperature Eluents A: H₂O + 0.1%formic acid, B: acetonitrile + 0.1% formic acid Flow Rate 1 ml/minGradient 50% B isocratic for 2.16 min

LCMS Method 6 Acquity LCTp2 Tof_Product Analysis Acidic

Instrument Waters Acquity UPLC with Waters LCT Premier XE detectorColumn Acquity BEH C18 1.7 μm 2.1 × 50 mm Column 50° C. TemperatureEluents A: H₂O + 0.1% formic acid, B: acetonitrile + 0.1% formic acidFlow Rate 1 ml/min Gradient 2%-98% B in 7.5 min

LCMS Method 7 SQ4 Acidic Polar

Instrument Waters Acquity UPLC with SQ detector Column Acquity BEH C181.7 μm 2.1 × 50 mm Column 50° C. Temperature Eluents A: H₂O + 0.1%formic acid, B: acetonitrile + 0.1% formic acid Flow Rate 1 ml/minGradient 1%-30% B in 1.20 min; 30%-98% B in 0.95 min

LCMS Method 8 SQ4 Acidic

Instrument Waters Acquity UPLC with SQ detector Column Acquity BEH C181.7 μm 2.1 × 50 mm Column 50° C. Temperature Eluents A: H₂O + 0.1%formic acid, B: acetonitrile + 0.1% formic acid Flow Rate 1 ml/minGradient 2%-98% B in 1.76 min

LCMS Method 9 IPC

Instrument Micromass Quattro micro API, Agilent 1100 Series pump ColumnXbridge BEH C18 2.5 μm 3.0 × 100 mm Column 80° C. Temperature Eluents A:H₂O + 200 mM HFIP + 8 mM TEA, B: methanol Flow Rate 1 ml/min Gradient30% B until 3.65 min; to 99% B at 3.75 min, 99% B until 3.85 min

LCMS Method 10 Scout Basic Peptide

Instrument Agilent 1100 HPLC; Waters Micromass ZQ Mass SpectrometerColumn Xbridge C18 3.5 μm, 30 × 3.0 mm, 3.5 μm Column 40° C. TemperatureEluents A: H₂O + 5 mM ammonium hydroxide, B: acetonitrile Flow Rate 2ml/min Gradient 5% to 80% B in 4.30 min, to 95% B at 5.0 min

Abbreviations:

-   ATP adenosine 5′-triphosphate-   Atm atmosphere-   AcOH acetic acid-   Aq aqueous-   Ar aryl-   br broad-   br.s., bs broad singlet-   BSA bovine serum albumin-   ° C. Celsius-   CDCl₃ deuterated chloroform-   CH₂Cl₂, DCM dichloromethane-   CH₃CN, MeCN acetonitrile-   d doublet-   dd doublet of doublets-   ddd doublet of doublets of doublets-   DIPEA N-ethyldiisopropylamine-   DMF N,N-dimethylformamide-   DMAP dimethyl aminopyridine-   DMSO dimethylsulfoxide-   dt doublet of triplets-   ESI electrospray ionization-   EtOAc ethyl acetate-   EtOH ethanol-   FCC flash column chromatography-   G gauge-   GMP guanosine 5′-monophosphate-   GDP guanosine 5′-diphosphate-   GTP guanosine 5′-triphosphate-   h hour-   HCl hydrochloric acid-   HATU    1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium    3-oxid hexafluorophosphate-   HBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid-   HFIP hexafluoro isopropanol-   HOBt hydoxybenzotriazole-   HPLC high pressure liquid chromatography-   i-PrOH isopropyl alcohol-   H₂O water-   K kelvin-   KOH potassium hydroxide-   LC liquid chromatography-   M molar-   m multiplet, mass-   MeOH methanol-   MES 2-(N-morpholino)ethanesulfonic acid-   MgSO₄ magnesium sulfate-   MHz megahertz-   mL milliliter-   mm millimeter-   mmol millimole-   min. minute-   mRNA messenger ribonucleic acid-   MS mass spectroscopy-   mw microwave-   m/z mass to charge ratio-   NaOH sodium hydroxide-   Na₂SO₄ sodium sulfate-   NEt₃ triethylamine-   NH₃ ammonia-   NMR nuclear magnetic resonance-   PBS phosphate buffered saline-   ppt precipitate-   ppm parts per million-   rbf round bottom flask-   Rf retardation factor-   RP reverse phase-   RT,rt room temperature-   Rt Retention time-   s singlet-   sat. saturated-   SM starting material-   t triplet-   TBA tributylamine-   TEA triethylamine-   TEAB triethylammonium bicarbonate-   TFA trifluoroacetic acid-   THF tetrahydrofuran-   TLC thin layer chromatography-   TRIS 2-Amino-2-hydroxymethyl-propane-1,3-diol-   UPLC ultra performance liquid chromatography-   Tris·HCl aminotris(hydroxymethyl)methane hydrochloride-   wt weight-   Xphos Pd G2 2^(nd) Generation Xphos Precatalyst,    Chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)-   μg microgram-   μL microliter

Nucleotide trialkylammonium salts were obtained from the correspondingcommercially available sodium salts following literature procedures (Y.Thillier, E. Decroly, F. Morvan, B. Canard, J.-J. Vasseur, F. Debart RNA2012, 18, 856-868; GMP Na-salt: Sigma-Aldrich No. G8377; GDP Na-salt:Sigma-Aldrich No. G7127; 2′-deoxy GMP Na-salt: Sigma-Aldrich No. D9500;Inosine 5′-monophosphate Na-salt: Sigma-Aldrich No. I4625; Inosine5′-diphosphate Na-salt: Sigma-Aldrich No. I4375) or under conditionssimilar to those described below.

GMP Triethylammonium Salt:

GMP sodium salt (2.0 g, 4.91 mmol) was dissolved RNase free water (40mL) and passed over DOWEX resin (50WX8, 40 g, rinsed thoroughly withH₂O) into an ice-cooled solution of triethylamine (10 g, 98 mmol) inEtOH (60 mL). The elution was followed by measuring UV absorption of theeluent, and the resin was rinsed with water until no further GMP eluted.The resulting solution was concentrated in vacuum to ca. 500 mL and thenlyophilized to obtain the title product as white solid (2.2 g, GMP/TEA1/2 judged by ¹H-NMR).

GDP Tributylammonium Salt:

GDP-disodium salt (1.17 g, 2.40 mmol) was dissolved water (100 mL) andpassed over DOWEX resin (50WX8, 10 mL, >1.7 meq/mL, rinsed thoroughlywith H₂O) into an ice-cooled solution of tributylamine (1.34 g, 7.20mmol) in EtOH (20 mL). The resin was rinsed with water (1000 mL) and theresulting solution was concentrated in vacuum to ca. 500 mL) and thenlyophilized to obtain the title product as colorless solid (1.88 g,GDP/TBA 1/2.5 judged by ¹H-NMR).

General Procedures for N⁷ Alkylation of GMP

Method A: In a 2-dram vial, guanosine monophosphate tributylammoniumsalt (50 mg, 68 umol) was dissolved in DMSO (680 uL) and treated withcommercially available alkyl bromide or chloride reagent (4 equiv.).When chloride alkylating reagents were used and in case of the(2-bromoethoxy)-benzene substrates, NaI was added (5 mg, 0.5 eq.). Thesolution obtained was shaken or stirred at 40° C. for 18 h. The solutionobtained was directly subjected to purification by HPLC (reversed phase,H₂O+0.1% TFA to MeCN+0.1% TFA 0-100%). Fractions that contained thedesired product were pooled, and the solvent was removed bylyophilzation to obtain the pure products as colorless solids or foams.

Method B: In a 2-dram vial, a 0.1 M solution of GMP or GDP triethyl- ortributylammonium salt in DMSO was treated with the bromide alkylatingreagent (4 equiv.). The solution was stirred at room temperature or 55°C. for 18 h and then directly subjected to purification by reversedphase column chromatography (ISCO Teledyne C18aq. Eluent: 0.1 Mtriethylammonium bicarbonate (pH=8.0) to MeCN 0-100%). Fractions thatcontained the desired product were pooled, and the solvent was removedby lyophilzation to obtain the pure products as tributylammonium saltsas colorless solids or foams.

Example 1. N⁷-([1,1′-Biphenyl]-4-ylmethyl)-5′-GMP TEA Salt

A solution of guanosine monophosphate triethylammonium salt (100 mg,0.177 mmol) and 4-biphenylmethyl bromide (175 mg, 0.710 mmol) in DMSO (1mL) was stirred at 55° C. overnight. The solution was directly subjectedto purification by reversed phase column chromatography (ISCO TeledyneC18 30 g Gold cartridge, Eluent A: 0.1 M TEAB; B: 20% MeCN in 0.1 MTEAB; Gradient: 0-100% B/A). The product fractions were pooled andlyophilized to give the title compound as white powder (38 mg, 28%). ¹HNMR (400 MHz, D₂O) δ ppm: 7.58-7.67 (4H, m), 7.46-7.53 (4H, m),7.38-7.46 (1H, m), 5.97-6.03 (1H, m), 5.63-5.76 (2H, m), 4.62-4.69 (1H,m), 4.46-4.52 (1H, m), 4.32-4.40 (1H, m), 3.97-4.18 (2H, m), 3.11-3.25(8.6H, q), 1.20-1.34 (13H, t). ³¹P NMR (162 MHz, D₂O): δ ppm: 3.73 (1P).LCMS method 2 R_(t): 1.39 min, MS [M+H]⁺ Observed: 529.8. Calculated:530.1.

Example 2. N⁷-([1,1′-Biphenyl]-4-ylmethyl)-5′-GDP TEA Salt

A solution of guanosine diphosphate triethylammonium salt (400 mg, 0.538mmol) and 4-biphenylmethyl bromide (400 mg, 1.619 mmol) in DMSO (2 mL)was stirred at room temperature overnight. 1M NaClO₄ acetone solution(3.23 mL) was added and then diluted with acetone. The precipitate wasseparated by centrifugation, washed with acetone and dried under vacuum.The solid thus obtained was dissolved in 0.1 M TEAB (5 mL). Theresulting solution was subjected to purification by reversed phasecolumn chromatography (ISCO, Teledyne C18, 30 g Gold cartridge, EluentA: 0.1 M TEAB; B: 90% MeCN in 0.1 M TEAB; Gradient: 0-100% B/A). Theproduct fractions were pooled and lyophilized to give the titledcompound as white powder (90 mg, 18%). ¹H NMR (400 MHz, D₂O) δ ppm:7.57-7.67 (4H, m), 7.39-7.53 (5H, m), 5.92 -6.00 (1H, d), 5.64-5.75 (2H,m), 4.67-4.72 (2H, m), 4.56-4.64 (1H, m), 4.34-4.40 (1H, m), 4.26-4.34(2H, m), 3.12-3.24 (12H, q), 1.27 (18H, t). ³¹P NMR (162 MHz, D₂O): δppm: 7.16 (1P), 10.93 (1P). LCMS method 2 R_(t): 1.43 min, MS [M−H]⁺observed: 608.2, calculated: 608.1.

Example 3. N⁷-(4-Chlorophenoxyethyl)-5′-GDP TEA Salt

A solution of guanosine diphosphate triethylammonium salt (500 mg, 0.672mmol) and 4-chlorophenyl 2-bromoethyl ether (633 mg, 2.69 mmol) in DMSO(4 mL) was stirred at 55° C. overnight. 1M NaClO₄ acetone solution (4mL) was added and then diluted with acetone. The precipitate wasseparated by centrifugation, washed with acetone and dried under vacuum.The solid thus obtained was dissolved in 0.1 M TEAB (5 mL). Theresulting solution was subjected to purification by ion exchangechromatography (TOSOH, TSKgel DEAE-5PW, 21.5 mm×15 cm, 13 μm, Eluent A:H₂O; B: 1M TEAB in water; Gradient: 0-100% B/A). The product fractionswere pooled and lyophilized to give the title compound as white powder(25 mg, 4%). ¹H NMR (400 MHz, D₂O) δ ppm: 7.22-7.31 (2H, d), 6.86-6.94(2H, d), 5.96-6.03 (1H, m), 4.46-4.52 (1H, m), 4.37-4.44 (1H, m),4.29-4.37 (1H, m), 4.19-4.29 (2H, m), 3.12-3.26 (9H, q), 1.20-1.34 (16H,t). ³¹P NMR (162 MHz, D₂O): δ ppm: 9.91 (1P), 11.28 (1P). LCMS method 2R_(t): 1.20 min, MS [M−H]⁺ observed: 596.8, calculated: 596.0.

Example 4. N⁷-(4-Chlorophenoxyethyl)-5′-GMP TEA Salt

Prepared as described previously (A. R. Kore et al Bioorg. Med. Chem.2013, 21, 4570; Chen et al J. Med. Chem. 2012, 55, 3837). The productwas obtained as white powder. (84 mg, 12%). ¹H NMR (400 MHz, D₂O) δ ppm:7.18-7.27 (2H, d), 6.83-6.93 (2H, d), 5.92-6.02 (1H, m), 4.41-4.48 (1H,m), 4.33-4.41 (1H, m), 4.11-4.20 (1H, m), 3.97-4.09 (1H, m), 3.13-3.26(10H, q), 1.20-1.34 (15H, t). ³¹P NMR (162 MHz, D₂O): δ ppm: 3.39 (1P).LCMS method 2 R_(t): 1.20 min, MS [M+H]⁺ observed: 517.8, calculated:518.1.

Example 5. N⁷-Benzyl-5′-GMP TEA Salt

Prepared as described previously (Brown et al J. Mol Biol. 2007, 372,7-15). The product was obtained as white powder (55 mg, 38%). ¹H NMR(400 MHz, D₂O) δ ppm: 7.33-7.45 (5H, m), 6.02-6.11 (1H, m), 5.64-5.73(2H, m), 4.44-4.53 (1H, m), 4.33-4.42 (1H, m), 3.96-4.17 (2H, m), 3.19(10H, q), 1.27 (14H, t). ³¹P NMR (162 MHz, D₂O): δ ppm: 3.78 (1P). LCMSmethod 2 R_(t): 0.84 min, MS [M+H]⁺ observed: 454.2, calculated: 454.1.

Example 6. N⁷-(6-phenylpyridin-3-yl)methyl)-5′-GMP TEA Salt

Step 1. 5-(bromomethyl)-2-phenylpyridine

A solution of (6-phenylpyridin-3-yl)methanol (300 mg, 1.620 mmol) in 33%HBr in acetic acid (2.93 mL, 16.20 mmol) was stirred at 40° C.overnight. The volatiles were evaporated. The residue was partitionedbetween DCM, diluted with sodium bicarbonate solution, and the organiclayer was separated. The organic layer was washed with water, dried, andconcentrated under vacuum to give a solid (277 mg, 69%). LCMS method 3R_(t): 1.36 min, MS [M+H]⁺ observed: 249.9, calculated: 250.0.

Step 2. N⁷-(6-phenylpyridin-3-yl)methyl)-5′-GMP TEA Salt

The title compound (40 mg, 23%) was prepared from 130 mg of GMP TEAsalts by the method described in Example 5. ¹H NMR (400 MHz, D₂O) δ ppm:8.57-8.66 (1H, m), 7.85-7.93 (1H, m), 7.66-7.82 (3H, m), 7.39-7.52 (3H,m), 5.91-6.02 (1H, m), 5.58-5.77 (2H, m), 4.42-4.50 (1H, m), 4.28-4.37(1H, m), 4.06-4.17 (1H, m), 3.95-4.02 (1H, m), 3.06-3.23 (10H, m),1.11-1.30 (15H, m). ³¹P NMR (162 MHz, D₂O): δ ppm: 3.54 (1P). LCMSmethod 2 R_(t): 0.91 min, MS [M+H]⁺ observed: 531.0, calculated: 531.1.

Example 7. N⁷-([1,1′-Biphenyl]-4-ylmethyl)-2′,3′-isopropylidene-5′-GMPTEA Salt

Step 1: N⁷-([1,1′-biphenyl]-4-ylmethyl)-2′,3′-isopropylidene guanosine

A solution of 2′,3′-isopropylidene guanosine (500 mg, 1.547 mmol) and4-biphenylmethyl bromide (145 mg, 0.849 mmol) in DMSO (2 mL) was stirredat room temperature overnight. The solution was directly subjected topurification by reversed phase HPLC (X-Bridge 50×50 mmm, 5 μm column,Eluent A: water with 5 mM NH₄OH; B MeCN with 5 mM NH₄OH; Gradient:15-40% B/A). The product fractions were pooled and lyophilized to givethe title compound as white powder (260 mg, 34%). LCMS method 1 R_(t):1.02 min, MS [M+H]⁺ observed: 490.1, calculated: 490.2.

Step 2: N⁷-([1,1′-Biphenyl]-4-ylmethyl)-2′,3′-isopropylidene-5′-GMP TEASalt

Phosphoryl trichloride (95 μL, 1.021 mmol) was added slowly to a mixtureof N⁷-([1,1′-biphenyl]-4-ylmethyl)-2′,3′-isopropylidene guanosine (100mg, 0.204 mmol) in trimethyl phosphate (1 mL) at 0° C., and stirredunder N₂ for 3 hrs. The reaction mixture was added dropwise to 1M TEABsolution (3 mL) at 0° C., the resulting mixture was centrifuged. Thesolution thus obtained was subjected to purification by reversed phasecolumn chromatography (ISCO Teledyne C18 30 g Gold cartridge, Eluent A:0.1 M TEAB; B: 30% MeCN in 0.1 M TEAB; Gradient: 0-100% B/A). Theproduct fractions were pooled and lyophilized to give the title compoundas white powder (38 mg, 24%). ¹H NMR (400 MHz, D₂O) δ ppm: 9.70-9.90(1H, br), 7.57-7.69 (4H, m), 7.29-7.57 (5H, m), 6.01-6.13 (1H, m),5.61-5.78 (2H, m), 5.34-5.41 (1H, m), 5.09-5.15 (1H, m), 4.52-4.60 (1H,m), 3.98-4.09 (1H, m), 3.80-3.92 (1H, m), 2.60 (8H, q), 1.50 (3H, s),1.34 (4H, s), 1.00 (14H, t). ³¹C (162 MHz, D₂O): δ ppm: 0.224 (1P). LCMSmethod 4 R_(t): 0.94 min, MS [M+H]⁺ observed: 570.1, calculated: 570.2.

Example 8.7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2-(phosphonooxy)ethoxy)methyl)-9H-purin-7-ium-6-olatetriethyl ammonium salt

Step 1:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2-hydroxyethoxy)methyl)-9H-purin-7-ium-6-olate

The alkylation of acycloguanosine (Sigma-Aldrich No. A4669) was carriedout following general procedure B. LC-MS method 1 R_(t)=0.95 mins; MSm/z [M+H]⁺ 392.2.

Step 2:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2-(phosphonooxy)ethoxy)methyl)-9H-purin-7-ium-6-olatetriethyl ammonium salt

The alcohol obtained in step 1 (73 mg, 0.19 mmol) was suspended inPO(OMe)₃ (1.9 mL), and POCl₃ (86 mg, 0.56 mmol) was added. The solutionwas stirred for 3 h at rt and then quenched by addition of TEAB (0.1 M,1 mL). The solution obtained was directly subjected to purification bycolumn chromatography (ISOC RP C18Aq eluting with TEAB 0.1M and MeCN;0-100% MeCN). After lyophilization of the fractions obtained, theproduct was obtained as colorless foam (84 mg, 0.14 mmol, 75%, ratiophosphate to NEt₃ 1/1.4 as determined by ¹H-NMR). LC-MS method 6R_(t)=1.79 mins; MS m/z [M+H]⁺ 472.1168; calculated: 472.1145, ¹H NMR(400 MHz, D₂O) δ 7.45 7.44 (3H, m), 7.54 (2H, d, J=8.14 Hz), 7.50 (2H,d, J=7.80 Hz), 5.53 (2H, s), 5.50 (2H, s), 3.78-3.74 (2H, m), 3.65-3.62(2H, m), 3.05 (8.4H, q, J=7.34 Hz, NEt₃), 1.13 (12.7H, t, J=7.56 Hz,NEt₃).

Example 9.7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2-((hydroxy(phosphonooxy)phosphoryl)oxy)ethoxy)methyl)-9H-purin-7-ium-6-olatetriethyl ammonium salt

-   Step 1: Acycloguanosine phosphate triethylammonium salt (obtained as    described above, 77 mg, 0.13 mmol) was suspended in DMF (anhydrous,    6.7 mL) and imidazole (101 mg, 1.48 mmol), 2,2′-dipyridyl disulfide    (151 mg, 0.685 mmol), and NEt₃ (34 mg, 0.24 mmol) were added. The    suspension was stirred for 10 min at rt before triphenylphosphine    (183 mg, 0.698 mmol) was added. The reaction mixture turned light    yellow and was stirred for 5 h at rt. NaClO₄ solution in acetone (1    M, 2.1 mL) and acetone (15 ml) were added and the suspension was    kept on ice for 10 min. The resulting solution was centrifuged (3    min, 2000×g) and the pellet was washed twice with 10 mL ice-cold    acetone. The pellet was dried under vacuum to obtain the imidazole    activated phosphate (50 mg, 0.096 mmol, 71%), which was used without    further purification.-   Step 2: The activated phosphate obtained in step 1 was suspended in    DMF (0.5 mL), and tributylammonium phosphate (1.0 M in DMF, 0.5 mL,    0.479 mmol) and ZnCl₂ (13 mg, 0.096 mmol) were added. The solution    was stirred vigorously at rt for 5 h and then directly subjected to    purification by column chromatography on ISCO RPAq18 eluting with    0.1 M TEAB and MeCN, 0 to 100% MeCN. Lyophilization of the product    containing fractions gave the title product as colorless solid (52    mg, 0.065 mmol, 68%, ratio of phosphate to NEt₃ 1/2.45 as determined    by ¹H NMR). LC-MS: R_(t)=0.99 mins; MS m/z [M+H]⁺ 552.1 LCMS method    1; ¹H NMR (400 MHz, D₂O) δ 7.59-7.54 (4H, m), 7.40-7.36 (4H, m),    7.32-7.28 (1H, m), 5.58 (2H, s), 5.55 (2H, s), 3.98-3.94 (2H, m),    3.73-3.71 (2H, m), 3.08 (q, J=7.22 Hz, NEt₃), 1.13 (12.7 H, t,    J=7.56 Hz, NEt₃).

Example 10. 7-((2-chloro-[1,1′-biphenyl]-4-yl)methyl)-5′-GDP TEA salt

Step 1: 2-chloro-4-methyl-1,1′-biphenyl

In a 2 dram vial, phenyl iodide (200 mg, 0.98 mmol) and(2-chloro-4-methylphenyl)boronic acid (167 mg, 0.98 mmol) were dissolvedin DMF (4.9 mL). An aquoues solution of K₂CO₃ (542 mg in 400 μL H₂O) andSphos Palladacycle G2 (7.1 mg, 9.8 μmol) was added, and the resultingsuspension was stirred vigorously at 80° C. for 3 h. After cooling toroom temperature, DCM (5 mL) was added, and the organic layer was washedwith water (10 mL), 10% LiCl (aq., 3×10 mL), and dried over Na₂SO₄. Theresidue obtained after removal of the solent in vacuo was subjected topurification by flash chromatography (SiO₂, heptane to 20% EtOAc inheptane). 2-Chloro-4-methyl-1,1′-biphenyl was obtained as colorless oil(147 mg, 0.725 mmol, 74%). ¹H NMR (400 MHz, CDCl₃) δ 7.45 7.44 (3H, m),7.41-7.36 (1H, m), 7.32 (1H, br s), 7.25 (1H, d, J=7.58 Hz), 7.14 (1H,d, J=7.58 Hz), 2.40 (3H, s).

Step 2: 4-(bromomethyl)-2-chloro-1,1′-biphenyl

2-Chloro-4-methyl-1,1′-biphenyl (140 mg, 0.691 mmol), N-bromosuccinimide (135 mg, 0.760 mmol), and azodiisobutyro nitrile (11 mg,0.069 mmol) were dissolved in CCl₄ (6.9 mL), and the solution wasstirred at 85° C. for 1 h. The solvent was removed under vacuum and theresidue was purified by flash chromatography (SiO₂, heptane to 20% EtOAcin heptane) to obtain the title compound in a mixture with startingmaterial (3:1, judged by 1H NMR) as colorless solid (173 mg, 0.406 mmol,66%). The mixture was used as such in the subsequent alkylationreaction.

Step 3: 7-((2-chloro-[1,1′-biphenyl]-4-yl)methyl)-5′-GDP TEA salt

GDP tributylammonium salt (70 mg, 0.086 mmol) was alkylated with theproduct of step 2 (73 mg, 0.26 mmol) following the general procedure B.The title product was obtained as colorless solid (30 mg, 0.036 mmol,42%). LCMS method 1: R_(t)=1.01 mins; MS m/z [M+H]⁺ 644.0; ¹H NMR (400MHz, DMSO-d₆) δ 10.42 (1H, s), 7.93 (1H, s), 7.78 (1H, d, J=8.12 Hz),7.72-7.63 (1H, m), 7.47-7.35 (9H, m), 5.81 (1H, s), 5.72 (1H, br s),4.48 (1H, br s), 4.40 (1H, br s), 4.14-4.04 (3H, m), 3.34 (br, NEt₃),1.03 (12.7H, t, J=7.56 Hz, NEt₃).

Example 11. 7-((3-methoxy-[1,1′-biphenyl]-4-yl)methyl)-5′-GDP TEA salt

Step 1: 3-methoxy-4-methyl-1,1′-biphenyl

In a 2 dram vial, phenyl iodide (200 mg, 0.98 mmol) and(3-methoxy-4-methylphenyl)boronic acid (163 mg, 0.98 mmol) weredissolved in DMF (4.9 mL). An aquoues solution of K₂CO₃ (542 mg in 400μL H₂O) and Sphos Palladacycle G2 (7.1 mg, 9.8 μmol) was added, and theresulting suspension was stirred vigorously at 80° C. for 12 h. Aftercooling to room temperature, DCM (5 mL) was added, and the organic layerwas washed with water (10 mL), 10% LiCl (aq., 3×10 mL), and dried overNa₂SO₄. The residue obtained after removal of the solent in vacuo wassubjected to purification by flash chromatography (SiO₂, heptane to 20%EtOAc in heptane). 3-Methoxy-4-methyl-1,1′-biphenyl was obtained ascolorless oil (149 mg, 0.752 mmol, 77%). ¹H NMR (400 MHz, DMSO-d6) δ7.70-7.66 (2H, m), 7.53-7.49 (2H, m), 7.44-7.39 (1H, m), 7.28 (1H, d,J=7.04 Hz), 7.19 (1H, dd, J=7.60, 1.77 Hz), 7.13 (1H, d, J=1.50 Hz),3.97 (3H, s), 2.37 (3H, s).

Step 2: 4-(bromomethyl)-3-methoxy-1,1′-biphenyl

3-methoxy-4-methyl-1,1′-biphenyl (147 mg, 0.741 mmol), N-bromosuccinimide (145 mg, 0.816 mmol), and azodiisobutyro nitrile (12 mg,0.074 mmol) were dissolved in CCl₄ (7.4 mL), and the solution wasstirred at 85° C. for 1 h. The solvent was removed under vacuum and theresidue was purified by flash chromatography (SiO₂, heptane to 20% EtOAcin heptane) to obtain the title compound as colorless solid (163 mg,0.588 mmol, 79%). ¹H NMR (400 MHz, CDCl₃) δ 7.62-7.59 (2H, m), 7.49-7.45(2H, m), 7.43-7.39 (2H, m), 7.18 (1H, dd, J=7.75, 1.66 Hz), 7.11 (1H, d,J=1.59 Hz), 4.65 (2H, s), 3.99 (3H, s).

Step 3: 7-((3-methoxy-[1,1′-biphenyl]-4-yl)methyl)-5′-GDP TEA salt

GDP tributylammonium salt (100 mg, 0.123 mmol) was alkylated with theproduct of step 2 (102 mg, 0.369 mmol) following the general procedureB. The title product was obtained as colorless solid (46 mg, 0.056 mmol,45%). LCMS method 1 R_(t)=0.99 mins; MS m/z [M+H]⁺ 640.0; ¹H NMR (400MHz, DMSO-d₆) δ 9.80 (1H, s), 7.66 (2H, d, J=7.49 Hz), 7.47-7.43 (3H,m), 7.38-7.34 (1H, m), 7.21 (1H, br s), 7.15 (1H, d, J=7.59 Hz), 5.83(1H, s), 5.56 (2H, s), 4.48 (2H, s), 4.14 (1H, br s), 4.06 (1H, br s),3.95 (3H, s), 2.68 (br, NEt₃), 1.04 (t, J=6.81 Hz, NEt₃).

Example 12. 7-(6-phenylpyridin-2(1H)-one-3-yl)-5′-GDP TEA salt

Step 1: 3-(hydroxymethyl)-6-phenylpyridin-2(1H)-one

To a solution of 2-oxo-6-phenyl-1,2-dihydropyridine-3-carboxylic acid(533 mg, 2.48 mmol) in THF (24 mL) was added borane dimethylsulfidecomplex (1.0 M in THF, 991 μL, 9.91 mmol), and the suspension wasstirred at room temperature for 18 h. MeOH was slowly added until gasevolution ceased. The resulting solution was partitioned between EtOAcand brine, and the organic layer was dried over NaSO₄. Removal of thesolvent under vacuum and purification of the residue by flashchromatography (SiO₂, DCM to 5% MeOH in DCM) gave the title compound aspale yellow solid (331 mg, 1.65 mmol, 66%). LCMS method 1 R_(t)=0.89mins; MS m/z [M−OH⁻]⁺ 183.6.

Step 2: 3-(bromomethyl)-6-phenylpyridin-2(1H)-one

3-(hydroxymethyl)-6-phenylpyridin-2(1H)-one (331 mg, 1.65 mmol) wastreated with HBr (aq. 48%, 16.5 mL), and the suspension was stirred atroom temperature for 1 h and then at 60° C. for 1 h. During this timethe suspension first clears, and then a precipitate is formed. Theprecipitate is filtered, washed thoroughly with water, and dried invacuum to give 3-(bromomethyl)-6-phenylpyridin-2(1H)-one as colorlesspowder (388 mg, 1.469 mmol, 89%). ¹H NMR (400 MHz, DMSO-d6) δ 7.08-7.74(4H, m), 7.55-7.51 (3H, m), 7.50-7.46 (1H, m), 5.28 (2H, s).

Step 3: 7-(6-phenylpyridin-2(1H)-one-3-yl)-5′-GDP TEA salt

GDP tributylammonium salt (100 mg, 0.123 mmol) was alkylated with theproduct of step 2 (102 mg, 0.369 mmol) following the general procedureB. The title product was obtained as colorless solid (46 mg, 0.056 mmol,45%). LCMS method 1 R_(t)=0.73 mins; MS m/z [M+H]⁺ 627.1; ¹H NMR (400MHz, DMSO-d₆) δ 9.7 (1H, s), 7.78-7.76 (2H, m), 7.63 (1H, d, J=7.33 Hz),7.47-7.55 (5H, m), 6.69 (1H, d, J=7.33 Hz), 5.82 (1H, d, J=2.04 Hz),5.45 (2H, s), 4.47 (1H, br), 4.40 (1H, dd, J=5.48 Hz), 4.13 (1H, br),4.08 (1H, br), 4.03 (1H, br), 3.35 (br, NEt₃), 1.05 (t, J=6.49 Hz,NEt₃).

Example 13. 7-(3,5-dimethylbenzyl)-5′-GDP TEA salt

GDP tributylammonium salt (150 mg, 0.184 mmol) was alkylated with3,5-dimethylbenzyl bromide (110 mg, 0.553 mmol) following the generalprocedure B. The title product was obtained as colorless solid (66 mg,0.090 mmol, 49%). LCMS method 1 R_(t)=0.80 mins; MS m/z [M+H]⁺ 561.9; ¹HNMR (400 MHz, DMSO-d₆) δ 10.38 (1H, s), 8.20 (1H, d, J=7.48 Hz), 7.95(1H, d, J=8.73 Hz), 7.65-7.58 (1H, m), 5.81 (2H, br s), 4.44 (1H, br s),4.38 (1H, br s), 4.13-4.04 (2H, m), 2.69 (br, NEt₃), 1.05 (t, J=6.49 Hz,NEt₃).

Example 14. 7-(3-fluorobenzyl)-guanosine-5′-GDP TEA salt

GDP tributylammonium salt (150 mg, 0.184 mmol) was alkylated with3-fluorobenzyl bromide (105 mg, 0.553 mmol) following the generalprocedure B. The title product was obtained as colorless solid (40 mg,0.055 mmol, 30%). LCMS method 1: R_(t)=0.59 mins; MS m/z [M+H]⁺ 551.9;¹H NMR (400 MHz, DMSO-d₆) δ 10.38 (1H, s),7.61 (1H, d, J=9.96 Hz), 7.54(1H, d, J=7.87 Hz), 7.38 (1H, dd, J=14.50, 7.44 Hz), 5.79 (1H, d, J=1.82Hz), 5.66 (1H, br s), 4.45 (1H, br s), 4.39 (1H, br s), 4.11-4.05 (2H,m), 2.73 (br, NEt₃), 1.05 (t, J=6.49 Hz, NEt₃).

Example 15. 7-((3-fluoro-[1,1′-biphenyl]-4-yl)methyl)-5′-GDP TEA salt

Step 1: 3-Fluoro-4-methyl-1,1′-biphenyl

In a 2 dram vial, phenyl iodide (800 mg, 3.92 mmol) and(3-fluoro-4-methylphenyl)boronic acid (604 mg, 3.92 mmol) were dissolvedin DMF (19.6 mL). An aquoues solution of K₂CO₃ (2.17 g in 1.9 mL H₂O)and Sphos Palladacycle G2 (28.3 mg, 39.0 μmol) was added, and theresulting suspension was stirred vigorously at 80° C. for 16 h. Aftercooling to room temperature, DCM (15 mL) was added, and the organiclayer was washed with water (10 mL), 10% LiCl (aq., 3×10 mL), and driedover Na₂SO₄. The residue obtained after removal of the solvent in vacuowas subjected to purification by flash chromatography (SiO₂, heptane to20% EtOAc in heptane). 3-Fluoro-4-methyl-1,1′-biphenyl was obtained ascolorless oil (746 mg, 3.61 mmol, 91%, 90% purity). ¹H NMR (400 MHz,DMSO-d6) δ 7.69-7.66 (2H, m), 7.48-7.36 (6H, m), 2.27 (3H, d, J=1.68Hz).

Step 2: 4-(bromomethyl)-3-fluoro-1,1′-biphenyl

3-Fluoro-4-methyl-1,1′-biphenyl (300 mg, 1.611 mmol), N-bromosuccinimide (315 mg, 1.77 mmol), and azodiisobutyro nitrile (26.5 mg,0.161 mmol) were dissolved in CCl₄ (16 mL), and the solution was stirredat 85° C. for 1 h. The solvent was removed under vacuum and the residuewas purified by flash chromatography (SiO₂, heptane to 20% EtOAc inheptane) to obtain the title compound as pale yellow solid (311 mg, 1.17mmol, 72%). ¹H NMR (400 MHz, DMSO-d6) δ 7.74-7.71 (2H, m), 7.62 (1H, d,J=7.92 Hz), 7.57 (1H, dd, J=11.47, 1.70 Hz), 7.53 (1H, dd, J=7.92, 1.60Hz), 7.51-7.46 (2H, m), 7.43-7.38 (1H, m).

Step 3: 7-((3-fluoro-[1,1′-biphenyl]-4-yl)methyl)-5′-GDP TEA salt

GDP tributylammonium salt (150 mg, 0.184 mmol) was alkylated with thebromide obtained in step 2 (147 mg, 0.553 mmol) following the generalprocedure B. The title product was obtained as colorless solid (45 mg,0.056 mmol, 30%). LCMS method 1 R_(t)=0.96 mins; MS m/z [M+H]⁺ 627.9; ¹HNMR (400 MHz, DMSO-d₆) δ 10.13 (1H, s), 7.69-7.67 (2H, m), 7.55-7.50(2H, m), 7.48-7.44 (3H, m), 7.41-7.38 (1H, m), 5.84-5.78 (2H, m),4.46-4.43 (2H, m), 4.11-4.06 (2H, m), 2.75 (br, NEt₃), 1.05 (t, J=6.49Hz, NEt₃).

Example 16. 7-(benzhydryl)-guanosine-5′-GDP TEA salt

GDP tributylammonium salt (150 mg, 0.184 mmol) was alkylated withbenzhydryll bromide (91 mg, 0.369 mmol) following the general procedureB. The title product was obtained as colorless solid (20 mg, 0.021 mmol,30%). LCMS method 1 R_(t)=0.90 mins; MS m/z [M+H]⁺ 610.1; ¹H NMR (400MHz, DMSO-d₆) δ 7.53-7.47 (2H, m), 7.44-7.29 (8H, m), 5.86 (1H, d,J=4.62 Hz), 5.82 (1H, d, J=4.32), 4.72 (1H, br s), 4.48-4.40 (1H, br s),4.20-4.02 (3H, m), 3.98-3.92 (2H, m), 2.62 (br, NEt₃), 1.00 (t, J=6.49Hz, NEt₃).

Example 17. 7-((1-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl4-yl)methyl)-5′-GDP TEA salt

Analytical data for N⁷-alkylated GMP derivatives are presented inTable 1. The following analogs were prepared similarly by generalmethods A or B or analogously to the methods described in Examples 1-17.

TABLE 1 Analytical data for N⁷-alkylated GMP derivatives.

Syn- Exp. thetic LCMS R_(t) Calc. mass Mass No. —Y—R¹ R³ R⁴ methodmethod min [M + H]⁺ [M + H]⁺ 1

H OH A 2 1.43 512.15 512.2 2

H OH A 7 0.71 514.1 514.5 3

H OH A 2 1.51 552.1 552.1 4

H OH A 2 1.23 498.1 498.4 5

H OH A 2 1.47 498.1 498.1 6

Me OH A 2 1.35′ 532.8 533.0 7

H OMe A 2 1.31′ 532.8 533.1 8

H OH A 2 1.12 502.1 502.2 9

H OH A 2 1.41 538.0 539.9 10

H OH B 2 1.17 579.2 579.8 11

H OH A 6 1.70′ 564.1290 564.1311 12

H OH A 6 1.62′ 546.1384 546.1390 13

H OH A 6 0.75′ 512.1177 512.1195 14

H OH A 6 0.72′ 472.1034 472.0985 15

H OH A 6 0.52' 479.1075 479.1103 16

H OH A 6 1.72' 637.1112 637.1117 17

H OH A 6 1.75′ 637.1112 637.1146 18

H OH A 6 1.68′ 560.1541 560.1544 19

H OH A 2 0.61 460.1 460.9 20

H OH A 2 0.68 445.0867 — 21

H OH A 6 0.64 499.0973 499.0975 22

H OH A 2 0.67 458.1 458.9 23

H OH A 6 0.80′ 484.1228 484.1217 24

H OH A 6 1.21′ 500.0999 500.0981 25

H OH A 6 1.06′ 480.1279 480.1281 26

H OH A 6 1.20′ 482.1435 482.1461 27

H OH A 6 0.91′ 578.1395 578.1382 28

H OH B 6 1.44′ 530.1435 530.1442 29

H OH A 2 1.42 535.1 535.1 30

H OH A 2 0.50 498.1 498.1 31

H OH A 2 1.05 512.1 512.1 32

H OH A 2 0.83 502.1 501.9 33

H OH A 6 1.20′ 522.0996 522.1002 34

H OH A 6 1.67′ 510.1748 510.1759 35

H OH A 6 0.90′ 522.0343 522.0334 36

H OH A 6 1.74′ 560.1541 560.1552 37

H OH A 6 1.30′ 538.0945 538.0955 38

H OH A 6 0.96′ 514.1334 514.1344 39

H OH A 6 1.20′ 608.1211 608.1221 40

H OH A 6 2.46′ 666.1960 666.1987 41

H OH A 6 2.05′ 642.0554 642.0544 42

H OH B 6 1.86′ 560.1541 560.1520 43

H OH B 6 1.03′ 547.1337 547.1345 44

H OH B 6 1.92′ 564.1046 564.1058 45

H OH B 6 1.77′ 548.1341 548.1339 46

H OH B 6 1.91′ 544.1592 544.1557 47

H OH A 8 0.73 548.1 548.3 48

H OH A 6 0.49′ 521.1293 521.1323 49

H OH A 1 1.09 536.1 536.3 50

H OH B 2 0.98' 520.1 519.9 51

H OH B 2 1.65' 536.2 535.9 52

H OH A 6 0.82′ 538.0904 538.0918 53

H OH A 6 1.34′ 558.1384 558.140 54

H OH A 2 1.28 537.1 537.0 55

H OH A 8 0.51 521.1 521.3 56

H OH A 6 1.42′ 555.1388 555.1341 57

H OH B 6 1.38′ 572.1541 572.1576 58

H OH A 2 1.00 536.1 536.0 59

H OH B 6 1.73′ 560.1541 560.1559 60

H OH A 2 1.31 536.1 536.1 61

H OH A 2 1.28 537.1 537.0 62

H H B 6 1.79′ 514.1486 514.1504 63

H OH A 8 0.69 536.1 536.3 64

H OH A 8 0.56 537.1 537.2 65

H OH A 2 1.30 511.1 511.2 66

H OH A 2 1.22 510.1 510.0 67

H OH A 6 0.66′ 496.0976 496.0986 68

H OH A 2 1.00 508.1 508.1 69

H OH A 6 0.74′ 508.1340 508.1321 70

H OH A 6 0.70′ 508.1340 508.1352 71

H OH A 6 0.63′ 508.1340 508.1350 72 Me H OH A 2 0.27 378.1 378.1 73

H OH B 6 1.09′ 480.1279 480.1273 74

H OH A 2 1.34 512.1 512.1 75

H OH A 2 1.48 531.1 531.0

Analytical data for N⁷-alkylated GDP derivatives are presented in Table2. The following analogs were prepared similarly by general methods A orB or analogously to the methods described in Examples 1-17.

TABLE 2 Analytical data for N⁷-alkylated GDP derivatives

Exp. Synthetic LC/MS R_(t) Calc. mass Mass No. —Y—R¹ R³ R⁴ method methodMin [M + H]⁺ [M + H]⁺ 1 Me H Me A 2 0.30′ 472.1 473.1 2

H H B 2 1.69 617.2 617.3 3

H H B 1 0.55 579.1 579.0 4

H H B 1 0.82 584.1 584.2 5

H H B 1 0.83 584.1 584.2 6

H H A 2 1.00 616.1 616.1 7

H H A 2 0.96 617.1 617.1 8

H H A 2 1.10 618.0 619.9 9

H H A 2 1.30 617.1 617.0 10

H H A 2 0.98 591.1 591.0 11

H H A 2 1.23 611.1 611.0 12

H H A 2 1.06 592.1 592.1 13

H H A 2 1.45 616.1 616.1 14

H H A 1 0.91′ 594.8 594.0

Analytical data for N⁷-alkylated ionosine derivatives are presented inTable 3. The following analogs were prepared similarly by generalmethods A or B or analogously to the methods described in Examples 1-17.

TABLE 3 Analytical data for N⁷-alkylated inosine derivatives.

Exp. Synthetic LC/MS Calc. mass Mass No. n method method R_(t) [M + H]⁺[M + H]⁺ 1 1 B 2 1.38 515.1 515.2 2 2 B 2 1.47 595.1 595.1

Procedures for Synthesis of C8 Substituted Xanthines Example 18.7-([1,1′-biphenyl]-4-ylmethyl)-8-bromo-3-methyl-1H-purine-2,6(3H,7H)-dione

A mixture of 8-bromo-3-methyl-1H-purine-2,6(3H,7H)-dione (245 mg, 1.000mmol), 4-(bromomethyl)-1,1′-biphenyl (247 mg, 1.000 mmol), and K₂CO₃(38.5 mg, 1.000 mmol) in DMF (5 mL) was stirred at RT overnight. Waterwas added to the reaction mixture. The resultant solid was collected byfiltration, washed with water, and dried. ISCO purification (Silica 80g, 0-5% MeOH in DCM) gave the titled product.

Example 19:(4-(7-([1,1′-biphenyl]-4-ylmethyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)phenyl)phosphonicacid

A reaction mixture of Example 18 (60 mg, 0.146 mmol), dimethyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)phosphonate (45.5mg, 0.146 mmol), Na₂CO₃ (30.9 mg, 0.292 mmol),tetrakis(triphenylphosphine)palladium (16.9 mg, 0.015 mmol), and water(0.2 mL) in dioxane (1 mL) was purged with N₂ for 2 minutes, thenstirred at 100° C. in a sealed vial overnight. Prep-HPLC purification(Waters X-Bridge C18 30×50 mm 5 um column, ACN/H₂O w/5 mM NH₄OH @75ml/min, 15-40% ACN over 3.5 min gradient) isolated mono-methyl ester anddimethyl ester. The fractions containing these two products werecombined and concentrated under vacuum. The residue was dissolved in DMF(1 mL), bromotrimethylsilane (112 mg, 0.729 mmol) was added. Thereaction mixture was stirred at RT overnight. Water and MeOH was addedto quench the reaction, and the resultant solution was concentratedunder vacuum. Prep-HPLC purification (Waters X-Bridge C18 30×50 mm 5 umcolumn ACN/H₂O w/5 mM NH₄OH @75 ml/min, 5-20% ACN over 3.2 min gradient)gave the desired product. ¹H NMR (400 MHz, DMSO-d6): δ ppm 7.64-7.77(2H, m), 7.54-7.64 (6H, m), 7.27-7.46 (3H, m), 7.03-7.15 (2H, m),5.56-5.71 (2H, m), 3.42 (3H, s). LCMS (XBridge C18 3.5 μm 3.0×30 mmACN/H₂O w/5 mM NH₄OH @75 ml/min, 5-95 over 2 min gradient) R_(t)=0.51min, MS [M+H]⁺ Observed: 489.3, calculated: 489.4.

Example 20:7-([1,1′-biphenyl]-4-ylmethyl)-8-bromo-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione

The titled product was prepared analogously according to the methoddescribed above. ¹H NMR (400 MHz, methanol-d4): δ ppm 7.88-8.00 (2H, m),7.68-7.78 (2H, m), 7.49-7.59 (4H, m), 7.27-7.44 (3H, m), 7.03-7.15 (2H,m), 5.72-5.81 (2H, m), 3.63 (3H, s), 3.38 (3H, s). LCMS (XBridge C18 3.5μm 3.0×30 mm ACN/H₂O w/5 mM NH₄OH @75 ml/min, 5-95 over 2 min gradient)Rt: 0.63 min, MS [M+H]⁺ observed: 503.3, calculated: 503.4.

General Synthesis of C8 Substituted Purines:

Step 1: 6-amino-5-((2-(4-chlorophenoxy)ethyl)amino)pyrimidin-4(3H)-one

2-(4-chlorophenoxy)acetic acid (13.3 g, 71.4 mmol) was dissolved in DMF(90 mL, 0.8 M), and triethylamine (14.4 g, 143 mmol, 2 equiv.) and HATU(27.1 g, 71.4 mmol) was added. The reaction was stirred at roomtemperature for 30 min before the pyridone (9.0 g, 71.4 mmol) was added.After stirring the mixture at room temperature over night, the reactionwas quenched with water, and the pH was adjusted to 1 by addition of 5 NHCl. The white precipitate was collected, washed with 1 N HCl, and driedunder vacuum for 2 days (12.7 g, 43.1 mmol, 60%). The amide obtained wasused as such in the next step.

The amide obtained above (10 g, 33.9 mmol) was dissolved in THF (250 mL)and cooled to 0° C. LiAlH₄ (7.73 g, 204 mmol) was slowly added, and themixture was stirred at room temperature for 30 min and 50° C. for 5hours. The mixture was then cooled to 0° C. and quenched by addition of1N HCl. The white precipitate was collected, washed with 1 N HCl, anddried under vacuum for 2 days. The product was obtained as white solidand was used without further purification (3.0 g, 10.7 mmol, 32%).

Step 2: General Procedure for Acylation and Ring Closure

The carboxylic acid was taken up in DMF (0.1 M) and treated with HATU (2equiv.) and triethylamine (4.5 equiv.). The solution was stirred at roomtemperature for 15 min before the pyrimidine obtained in step 1 wasadded. The resulting solution was stirred for 16 h at room temperatureand then diluted with EtOAc and washed with a buffered solution(pH=7.0). The aqueous layers were extracted 2× with EtOAc, and thecombined organic layers were dried over Na₂SO₄. Concentration of thesolution under vacuum gave a brown oil, which was purified by RP-HPLC.

-   For R═OH: The amide obtained above was taken up in isopropanol (0.1    M), sodium tert-butoxide was added (5 equiv.), and the mixture was    stirred at 80° C. for 4 h. Acetic acid was added until the solution    was acidic and a precipitate was formed. The precipitate was    filtered, washed with water, and dried in vacuum. The resulting    alcohol was phosphorylated by treatment with POCl₃ in    trimethylphosphate for 2 h at 0° C. The reaction was quenched with    TEAB (1M) and directly subjected to purification by RP-HPLC.-   For R═PO(OEt)₂: The phosphate esters obtained above were hydrolyzed    by treatment of a 0.7 M solution in DMF with TMSBr (5.5 eq.). After    stirring for 16 h at room temperature, the reaction was quenched by    addition of MeOH/H₂O 1/1 and 1 M TEAB. The suspension was filtered    and directly subjected to purification by RP-HPLC.

Analytical data for C8-substituted purines are presented in Table 4. Thefollowing analogs were prepared by methods described above.

TABLE 4 Analytical data for C8 substituted purines.

Calc. Exp. LC/MS mass Mass No. Z₂ method R_(t) [M + H]⁺ [M + H]⁺ 3

1 0.95′ 447.8 447.1 4

2 1.44′ 525.8 (M − H⁺) 525.3 (M − H⁺) 5

1 1.59′ 463.8 463.3 6

1 0.91′ 413.8 413.1 7

10  1.64′ 469.8 469.3 8

3 0.14′ 441.8 441.2

Preparation of Imidazole Activated mRNA Caps

General Procedure for Imidazole Activation for GMP and GDP Derivatives:

To a solution of the triethylammonium salt of the N⁷-alkylated-GMP/GDPderivative in DMF was added imidazole (10 eq.), 2,2′-dipyridyl disulfide(4 eq.) and TEA (2 eq.). The reaction stirred at RT for 5 min and thentriphenylphosphine (4 eq.) was added turning the reaction yellow incolor. After stirring at RT for 16-18 h, 1M NaClO₄ in acetone (10 eq.)was added to the reaction generating a white precipitate. Additionalacetone was added to the reaction, and the resulting suspension wascooled at 4° C. for 20 min and centrifuged. The supernatant was decantedand the precipitate washed with acetone, cooled at 4° C. for 10 min, andcentrifuged. This wash procedure was repeated one or two more times. Theresulting precipitate was dried under vacuum to give the sodium salt ofthe imidazole activated N⁷-alkylated GMP/GDP derivative.

Example 21: P²-imidazolide N⁷-([1,1′-biphenyl]-4-ylmethyl)-5′-GDP Nasalt

A mixture of triethylammonium salt of N⁷-([1,1′-biphenyl]-4-ylmethyl)-5′GDP (45 mg, 0.049 mmol), imidazole (50 mg, 0.742 mmol),2,2′-dithiodipyridine (65 mg, 0.297 mmol), triethylamine (207 μL, 1.484mmol), and triphenylphosphine (78 mg, 0.297 mmol) in dimethylformamide(1.0 mL) was stirred at room temperature for 3 h. A solution of sodiumperchlorate in acetone (1M, 1 mL) was added and then diluted withacetone (40 mL). The precipitate was separated by centrifugation, washedwith acetone three times and dried under vacuum to afford the titlecompound as sodium salt (34 mg, 93%). ¹H NMR (400 MHz, D₂O) δ ppm:7.80-7.90 (1H, s), 7.63-7.72 (4H, m), 7.38-7.56 (5H, m), 7.18-7.27 (1H,s), 6.86-6.95 (1H, s), 5.97-6.07 (1H, m), 5.59-5.75 (2H, m), 4.58-4.64(2H, m), 4.33-4.40 (3H, m), 4.16-4.29 (1H, m), 4.06-4.16 (1H, m). ¹³CNMR(162 MHz, D₂O): δ ppm: 11.712 (1P), 19.863 (1P). LCMS method 2 R_(t):1.35 min, MS [M−H]⁺ observed: 659.1, calculated: 659.1.

Example 22: P²-imidazolide N⁷-([1,1′-biphenyl]-4-ylmethyl)-5′-GMP Nasalt

The title compound was synthesized analogously to the method describedin Example 21. ¹H NMR (400 MHz, D₂O) δ ppm: 7.78-7.83 (1H, s), 7.43-7.49(4H, m), 7.16-7.40 (5H, m), 7.01-7.08 (1H, s), 6.83-6.90 (1H, s),5.79-5.87 (1H, m), 5.51-5.76 (2H, m), 4.50-4.57 (1H, m), 4.20-4.35 (2H,m), 4.00-4.16 (2H, m). ³¹P NMR (162 MHz, D₂O): δ ppm: 8.134 (1P). LCMSmethod 2 R_(t): 1.32 min, MS [M−H]⁺ observed: 577.9, calculated: 578.2.

Example 23:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2-((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)ethoxy)methyl)-9H-purin-7-ium-6-olate

The title compound was synthesized following the general procedure forimidazole activation of bisphosphates described above. LCMS method 5 MS[M−H]⁺ observed: 602.3, calculated: 602.1.

Example 24:P²-imidazolide-N⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-5′-GDP Na salt

Step 1: N⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-guanosine

2′OMe-Guanosine (0.673 mmol) and 1-(2-bromoethoxy)-4-chlorobenzene (2.70mmol) were taken up in 4 ml anh. DMSO and stirred at 50° C. for 20 h.The reaction was cooled to RT, diluted with DMSO, and purified onprepatory HPLC [Shimadzu Prep HPLC (Sunfire Prep C18 5 μm, 100 mm×30 mmcolumn; 0-20 min: 5 to 30% 0.1% TFA in ACN/0.1% TFA in H₂O; 42 mL/min)].Product fractions were pooled and concentrated while azeotroping withtoluene to afford the trifluoroacetate salt of the title compound as awhite solid (99 mg, 0.17 mmol, 26%). LCMS method 2 R_(t)=1.38 mins; MSm/z [M+H]⁺ 452.2.

Step 2: N⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-5′-GMP TEA salt

To a solution of N⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-guanosine(0.085 mmol) in 1.0 ml trimethyl phosphate cooled to 0° C. was slowlyadded POCl₃ (0.536 mmol) dropwise. The reaction stirred at 0° C. for 6.5h and was then cooled to −20° C. for 16 h. The reaction was then warmedto 0° C. for 2 h. Additional POCl₃ (0.536 mmol) was added to thereaction and the reaction stirred at 0° C. for 3 h. The reaction wasslowly quenched at 0° C. with 1M TEAB(aq.) (3.5 ml). Upon completion ofthe TEAB addition, the reaction was warmed to RT to ensure quenching ofexcess POCl₃. The quenched reaction was purified directly on prepatoryion exchange chromatography [(Tosoh TSKgel DEAE-5PW, 13 μm, 21.5×150 mm,0-30 min: 0-100% 1M TEAB, 5 ml/min)]. Lyophilization of productfractions yielded the triethylammonium salt of the title compound as awhite solid, which was used directly in the next step. LCMS method 2R_(t)=1.34 mins; MS m/z [M+H]⁺ 532.9; ¹H NMR (D₂O) δ: 7.16-7.22 (m, 2H),6.80-6.86 (m, 2H), 6.03 (d, J=3.4 Hz, 1H), 4.78-4.84 (m, 2H), 4.48-4.54(m, 2H), 4.46 (t, J=5.3 Hz, 1H), 4.25-4.30 (m, 1H), 4.18-4.22 (m, 1H),4.09-4.17 (m, 1H), 3.96-4.03 (m, 1H), 3.51 (s, 3H), 3.14 (q, J=7.4 Hz,NEt₃), 1.21 (t, J=7.3 Hz, NEt₃).

Step 3: P¹-imidazolide N⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-5′-GMPNa salt

To a suspension of N⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-5′-GMP(0.05 mmol) in 1.0 ml anhydrous DMF was added imidazole (0.514 mmol),2,2′-dipyridyl disulfide (0.227 mol) and TEA (0.072 mmol). PPh₃ (0.229mmol) was then added to the reaction turning it yellow in color. Afterstirring at RT for 18 h, 1M NaClO₄ in acetone (0.250 mmol) was added tothe reaction followed by 4 ml of acetone. The resulting suspension wascooled at 4° C. for 20 min and centrifuged. The supernatant was decantedand the resulting precipitate was washed with 5 ml of acetone, cooled at4° C. for 20 min and centrifuged again. This wash procedure was repeatedtwo more times. The resulting off-white solid was dried under vacuum toafford the sodium salt of the title compound (30 mg). This compound wasused directly in Step 4. LCMS method 5 MS(ES⁻):m/z=580.4 (M−H⁺).

Step 4: N⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-5′-GDP TEA salt

To a solution of P¹-imidazolideN⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-5′-GMP Na salt (0.05 mmol) in1 ml anh. DMF was added tributylammonium orthophosphate (prepared asdescribed in: A. R. Kore, G. Parmar, Syn. Comm. 2006, 36, 3393-3399; 1 Min DMF, 0.250 mmol) dropwise followed by ZnCl₂ (0.051 mmol). Thereaction stirred at RT for 4.5 h and was then quenched with water (1 ml)and concentrated to an oily solid. This solid was taken up in DMF (1 ml)and 1 M NaClO₄ in acetone (0.500 mmol) was added followed by 7 ml ofacetone. The resulting suspension was cooled to 0° C. for ˜10 min,centrifuged, and the supernatant decanted. The solid was washed oncewith acetone, centrifuged, and the supernatant decanted. The resultingsolid was then dried under vacuum to remove any residual organics andtaken up in H₂O and purified using prepatory ion exchange chromatography[(Tosoh TSKgel DEAD-5PW, 13 μm, 21.5×150 mm, 0-100% 1M TEAB, 5 ml/min)].Product fractions were pooled, concentrated, and azeotroped with ethanolto afford the triethylammonium salt of the title compound as a whitesolid (5.7 mg, 6.6 μmol, 13%). LCMS method 5 MS(ES⁺):m/z=612.0 (M+H⁺);¹H NMR (D₂O) δ: 9.25 (s, 1H), 7.13 (d, J=8.9 Hz, 2H), 6.79 (d, J=8.9 Hz,2H), 5.99 (d, J=3.1 Hz, 1H), 4.76-4.87 (m, 2H), 4.40-4.54 (m, 3H),4.24-4.35 (m, 2H), 4.13-4.23 (m, 2H), 3.50 (s, 3H), 3.13 (q, J=7.3 Hz,NEt₃), 1.17-1.23 (m, J=7.3, 7.3 Hz, NEt₃).

Step 5: P²-imidazolide-N⁷-(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-5′-GDPNa salt

To a suspension of N⁷(2-(4-chlorophenoxy)ethyl)-2′-O-methyl-5′-GDP TEAsalt (6.55 μmol) in 0.5 ml anh. DMF was added imidazole (88 μmol),2,2′-dipyridyl disulfide (27.2 μmol) and TEA (14.3 μmol). PPh₃ (26.7μmol) was then added to the reaction turning it yellow in color. Afterstirring at RT for 18 h, 1 M NaClO₄ in acetone (0.100 mmol) was added tothe reaction followed by acetone (2 ml) generating a white precipitate.This suspension was cooled at 4° C. for 20 min and centrifuged. Thesupernatant was decanted and the resulting precipitate was washed withacetone (3 ml), cooled at 4° C. for 10 min and centrifuged. This acetonewash procedure was repeated one more time. The resulting white solid wasdried under vacuum to give the sodium salt of the title compound (4.6mg, 6.4 μmol, 97%). This compound was used directly in the mRNA cappingreaction. LCMS method 5 MS(ES⁻):m/z=660.2 (M−H⁺).

Example 25:P²-imidazolide-N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-GDP Na salt

Step 1: N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-guanosine

3′OMe-Guanosine (0.673 mmol) and 1-(2-bromoethoxy)-4-chlorobenzene (2.70mmol) were taken up in 4 ml anh. DMSO and stirred at 50° C. for 20 h.The reaction was cooled to RT, diluted with DMSO, and purified onprepatory HPLC [Shimadzu Prep HPLC (Sunfire Prep C18 5 μm, 100 mm×30 mmcolumn; 0-20 min: 5 to 30% 0.1% TFA in ACN/0.1% TFA in H₂O; 42 mL/min)].Product fractions were pooled and concentrated while azeotroping withtoluene to afford the triflouroacetate salt of the title compound as awhite solid (124 mg, 0.223 mmol, 33%). LCMS method 2 R_(t)=1.39 mins; MSm/z [M+H]⁺ 452.9.

Step 2: N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-GMP TEA salt

To a solution of N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-guanosine(0.115 mmol) in 1.0 ml trimethyl phosphate cooled to 0° C. was slowlyadded POCl₃ (0.536 mmol) dropwise. The reaction stirred at 0° C. for 6.5h and was then cooled to −20° C. for 16 h (placed in the freezerovernight). The reaction was then warmed to 0° C. for 2 h. AdditionalPOCl₃ (0.536 mmol) was added to the reaction and the reactions stirredat 0° C. for 3 h. The reaction was slowly quenched at 0° C. with 1MTEAB(aq.) (3.5 ml). Upon completion of the TEAB addition, the reactionwas warmed to RT to ensure quenching of excess POCl₃. The quenchedreaction was purified directly on prepatory ion exchange chromatography[(Tosoh TSKgel DEAE-5PW, 13 μm, 21.5×150 mm, 0-30 min: 0-100% 1M TEAB, 5ml/min)]. Lyophilization of product fractions yielded thetriethylammonium salt of the title compound as a white solid, which wasused as obtained in the next step. LCMS method 2 R_(t)=1.34 mins; MS m/z[M+H]⁺ 532.8; ¹H NMR (D₂O) δ: 7.16-7.23 (m, 2H), 6.79-6.87 (m, 2H), 5.94(d, J=4.0 Hz, 1H), 4.78-4.86 (m, 2H), 4.47-4.52 (m, J=5.0, 5.0 Hz, 2H),4.36-4.43 (m, 1H), 4.09-4.17 (m, 1H), 4.07 (t, J=5.0 Hz, 1H), 3.93-4.01(m, 1H), 3.43 (s, 3H), 3.13 (q, J=7.4 Hz, NEt₃), 1.21 (t, J=7.3 Hz,NEt₃).

Step 3: P¹-imidazolide-N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-GMPNa salt

To a suspension of N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-GMP TEAsalt (0.075 mmol) in 1.0 ml anh. DMF was added imidazole (0.734 mol),2,2′-dipyridyl disulfide (0.340 mmol) and TEA (0.108 mmol). PPh₃ (0.343mmol) was then added to the reaction turning it yellow in color. Afterstirring at RT for 18 h, 1M NaClO₄ in acetone (0.375 mmol) was addedfollowed by 4 ml of acetone. The resulting suspension was cooled at 4°C. for 20 min and centrifuged. The supernatant was decanted and theresulting precipitate was washed with acetone (5 ml), cooled at 4° C.for 20 min, and centrifuged. This wash procedure was repeated two moretimes. The resulting off-white solid was dried under vacuum to give thesodium salt of the title compound (45 mg). This compound was useddirectly in Step 4. LCMS method 5 MS(ES⁻):m/z=580.4 (M−H⁺).

Step 4: N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-GDP TEA salt

To a solution ofP¹-imidazolide-N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-GMP Na salt(0.075 mmol) in 1 ml anh. DMF was added tributylammonium orthophosphate(prepared as described in: A. R. Kore, G. Parmar, Syn. Comm. 2006, 36,3393-3399; 1 M in DMF, 0.375 mmol) dropwise followed by ZnCl₂ (0.076mmol). The rxn stirred at RT for 4.5 h and was then quenched with water(1 ml) and concentrated to an oily solid. This solid was taken up in DMF(1 ml) and 1M NaClO₄ in acetone (1.00 mmol) was added followed byacetone (7 ml). The resulting suspension was cooled to 0° C. for 10 min,centrifuged, and the supernatant decanted. The solid was washed oncewith acetone, centrifuged, and the supernatant decanted. The resultingsolid was then dried under vacuum to remove any residual organics andpurified using prepatory ion exchange chromatography [(Tosoh TSKgelDEAD-5PW, 13 μm, 21.5×150 mm, 0-100% 1M TEAB, 5 ml/min)]. Productfractions were pooled, concentrated, and azeotroped with ethanol toafford the triethylammonium salt of the title compound as a white solid(14.3 mg, 4.21 μmol, 8%). LCMS method 5 MS(ES⁻):m/z=610.2 (M−H⁺); ¹H NMR(D₂O) δ: 9.30 (s, 1H), 7.15 (d, J=9.0 Hz, 2H), 6.80 (d, J=9.0 Hz, 2H),5.92 (d, J=4.0 Hz, 1H), 4.77-4.87 (m, 2H), 4.71-4.75 (m, 1H), 4.39-4.49(m, 3H), 4.23-4.32 (m, 1H), 4.06-4.17 (m, 2H), 3.43 (s, 3H), 3.13 (q,J=7.4 Hz, NEt₃), 1.21 (t, J=7.3 Hz, NEt₃).

Step 5: P²-imidazolide-N⁷-(2-(4-chlorophenoxy)ethyl)-3′-O-methyl-5′-GDPNa salt

To a suspension of2-amino-7-(2-(4-chlorophenoxy)ethyl)-9-((2R,3R,4S,5R)-3-hydroxy-5-(((hydroxy(phosphonooxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium(4.20 μmol) in 0.5 ml anhydrous DMF was added imidazole (88 μmol),2,2′-dipyridyl disulfide (27.2 μmol) and TEA (14.3 μmol). PPh₃ (26.7μmol) was then added to the reaction turning it yellow in color. Afterstirring at RT for 18 h, 1M NaClO₄ in acetone (0.100 mmol) was added tothe reaction followed by acetone (2 ml) generating a white precipitate.This suspension was cooled at 4° C. for 20 min and centrifuged. Thesupernatant was decanted and the resulting precipitate was washed withacetone (3 ml), cooled at 4° C. for 10 min. and centrifuged. Thisacetone wash procedure was repeated one more time. The resulting whitesolid was dried under vacuum to give the sodium salt of the titlecompound (7.2 mg). This compound was used directly in the mRNA cappingreaction. LCMS method 5 MS(ES⁻):m/z=660.2 (M−H⁺).

Example 26:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)methyl)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium

Step 1: N⁷-([1,1′-biphenyl]-4-ylmethyl)-guanosine

Guanosine (2.65 mmol) and 4-bromomethylbiphenyl (6.07 mmol) were takenup in anhydrous DMSO (7.5 ml) and stirred at RT for 60 h. The reactionwas diluted with DMSO and purified on prepatory RP-HPLC. Pooled productfractions were concentrated while azeotroping with toluene, taken up in1:1 H₂O/acetonitrile, frozen, and lyophilized to give thetrifluoroacetate salt of the title compound as a white solid. LCMSmethod 1 R_(t)=0.94 mins; MS m/z [M+H]⁺ 450.0.

Step 2:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphonomethyl)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium

To a solution of N⁷-([1,1′-biphenyl]-4-ylmethyl)-guanosine (0.266 mmol)in 1.0 ml of trimethyl phosphate at −10° C. was slowly added a solutionof methylenediphosphonic dichloride (1.00 mmol) in 1.0 mltrimethylphosphate cooled to −10° C. The reaction stirred at −10° C.-0°C. for 6.5 h and then sat at −20° C. for 60 h. The reaction was slowlyquenched by adding it to 1M TEAB (9 ml) at 0° C. Upon completion of theaddition, the reaction was warmed to RT to ensure quenching of theexcess methylenediphosphonic dichloride. The resulting solution waspurified directly on prepatory HPLC [(Phenomenex Gemini-NX 5 μm C18 100mm×30 mm column; 0-15 min: 10 to 40% ACN/0.1M TEAB in H₂O, 25 ml/min)].Product fractions were pooled, frozen, and lyophilized to afford thetriethylammonium salt of the title compound as a white solid (16 mg,0.02 mmol, 7%). LCMS method 2 R_(t)=1.46 mins; MS m/z [M+H]⁺ 608.1; ¹HNMR (D₂O) δ: 9.51 (s, 1H), 7.25-7.35 (m, 4H), 7.10-7.25 (m, 5H), 5.70(d, J=3.4 Hz, 1H), 5.42-5.58 (m, 2H), 4.50 (t, J=3.9 Hz, 1H), 4.40 (t,J=5.1 Hz, 1H), 4.28-4.33 (m, 1H), 4.20-4.28 (m, 1H), 4.06-4.17 (m, 1H),3.12 (q, J=7.3 Hz, NEt₃), 2.17 (t, J=19.7 Hz, 2H), 1.20 (t, J=7.3 Hz,NEt₃).

Step 3:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)methyl)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium

To a solution of7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphonomethyl)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium(0.016 mmol) in 1.0 ml of anhydrous DMF was added imidazole (0.176mmol), 2,2′-dipyridyl disulfide (0.082 mmol) and TEA (0.029 mmol). Thereaction stirred at RT for 10 min and then triphenylphosphine (0.084mmol) was added turning the reaction yellow in color. After stirring atRT for 18 h, 1 M NaClO₄ in acetone (0.250 mmol) was added to thereaction followed by acetone (5 ml) generating a white precipitate. Theresulting suspension was cooled at 4° C. for 20 min and centrifuged. Thesupernatant was decanted and the precipitate washed with acetone (5 ml),cooled at 4° C. for 10 min, and centrifuged. This wash procedure wasrepeated one more time. The resulting precipitate was dried under vacuumto afford the sodium salt of the title compound as a white solid (10.8mg, 0.013 mmol, 81%). This compound was used directly in the mRNAcapping reaction. LCMS method 5 MS(ES⁻):m/z=657.5 (M−H⁺).

Example 27:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)amino)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium

Step 1:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphonoamino)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium

N⁷-([1,1′-biphenyl]-4-ylmethyl)-guanosine (0.177 mmol) was added to asolution of dichlorophosphinylphosphorimidic trichloride (J. Emsley, J.Moore, P. B. Udy, J. Chem. Soc. A 1971, 2863-2864; 0.891 mmol) intrimethylphosphate (2.0 ml) at 0° C. The reaction stirred at 0° C. for1.5 h and was then cooled to −20° C. and sat for 16 h. The reaction waswarmed to 0° C. and additional dichlorophosphinylphosphorimidictrichloride (0.891 mmol) was added to the reaction. The reaction stirredat 0° C. for 5 h until only traces of the starting material remained asmonitored by LCMS (Method 2 RXNMON_Acidic_Polar_PosNeg.olp-ZQ1). Thereaction was quenched by slowly adding it to 1M TEAB (10 ml) at 0° C.Upon completion of the addition, the reaction was warmed to RT to ensurequenching of the excess dichlorophosphinylphosphorimidic trichloride.The resulting suspension was filtered and the filtrate subjected topreparatory HPLC [(Phenomenex Gemini-NX 5 μm C18 100 mm×30 mm column;0-15 min: 10 to 50% ACN/0.1M TEAB in H₂O, 25 ml/min)]. Product fractionswere pooled, frozen, and lyophilized to the triethylammonium salt of thetitle compound as a white solid (13.5 mg, 0.018 mmol, 10%). LCMS method6 Calc.: 608.1186; Found: 609.1290 [M+H⁺]; ¹H NMR (D₂O) δ: 9.51 (s, 1H),7.49-7.57 (m, 4H), 7.38-7.44 (m, 4H), 7.31-7.38 (m, 1H), 5.90 (d, J=3.6Hz, 1H), 5.55-5.68 (m, 2H), 4.63 (t, J=4.3 Hz, 1H), 4.48 (t, J=5.1 Hz,1H), 4.29-4.38 (m, 1H), 4.17-4.26 (m, 1H), 4.04-4.17 (m, 1H), 3.13 (q,J=7.3 Hz, NEt₃), 1.21 (t, J=7.3 Hz, NEt₃).

Step 2:7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)amino)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium

7-([1,1′-biphenyl]-4-ylmethyl)-2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphonoamino)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-7-ium(0.007 mmol) was taken up in 1.0 ml of anh. DMF and imidazole (0.073mmol), 2,2′-dipyridyl disulfide (0.036 mmol) and TEA (0.014 mmol) wasadded. The reaction stirred at RT for 5 min and then triphenylphosphine(0.038 mmol) was added turning the reaction yellow in color. Afterstirring at RT for 16 h, 1M NaClO₄ in acetone (0.100 mmol) was added tothe reaction followed by acetone (5 ml) generating a white precipitate.The resulting suspension was cooled at 4° C. for 20 min and centrifuged.The supernatant was decanted and the precipitate washed with acetone (5ml), cooled at 4° C. for 10 min, and centrifuged. This wash procedurewas repeated one more time. The resulting precipitate was dried undervacuum to give the sodium salt of the title compound as a white solid(2.3 mg, 3.4 μmol, 33%). This compound was used directly in the mRNAcapping reaction. LCMS method 5 MS(ES⁻):m/z=658.3 (M−H⁺).

Analytical data for imidazole activated GDP derivatives are presented inTable 5. The following examples were prepared according to the generalprocedure for imidazole activation described above or analogously to themethods described in Example 21-27.

TABLE 5 Analytical data for imidazole activated GDP derivatives.

Exp. LC/MS Calc. mass Mass No. —Y—R¹ R³ R⁴ R² method R_(t) [M − H]⁻ [M −H]⁻ 1 Me H H NH₂ 5 — 507.3 507.3 2 Me H Me NH₂ 5 — 521.3 521.4 3

H H NH₂ 2 1.14′ 645.8 646.1 4

Me H NH₂ 5 — 661.9 661.4 5

H Me NH₂ 5 — 661.9 661.4 6

H H NH₂ 5 — 676.1 676.4 7

H H NH₂ 5 — 688.1 688.4 8

H H NH₂ 5 — 600.1 600.3 9

H H NH₂ 5 — 610.1 610.3 10

H H NH₂ 5 — 692.1 692.3 11

H H NH₂ 5 — 658.1 658.4 12

H H NH₂ 5 — 675.1 675.4 13

H H NH₂ 2 1.00 666.3 666.1 14

H H NH₂ 2 1.06 669.9 668.0 15

H H NH₂ 2 1.16 661.1 661.1 16

H H NH₂ 2 1.36 666.1 666.1 17

H H NH₂ 2 1.01 642.1 642.1 18

H H H 2 1.32′ 644.2 644.1

Analytical data for imidazole activated GMP derivatives are presented inTable 6. The following examples were prepared according to the generalprocedure for imidazole activation described above or analogously to themethods described in Example 21-27.

TABLE 6 Analytical data for imidazole activated GMP derivatives.

Calc. Exp. LC/MS mass Mass No. —Y—R¹ R³ R⁴ method R_(t) [M + H]⁺ [M +H]⁺ 1

H H 2 1.20′ 567.1 567.9 2

H Me 2 1.20′ 582.9 532.9 3

Me H 2 1.18′ 582.9 582.9 4

H H 2 0.85′ 505.1 505.2 5

H H 2 1.32′ 578.5 577.9

For capping of oligonucleotides and RNAs, the imidazole activated mono-and diphosphates, obtained in the examples shown above, were dissolvedin RNAse-free water to give a 20 mM solution of the imidazole activatedGMP/GDP derivative in H₂O (based on the amount of activated imidazolepresent in the sample as determined by ¹H-NMR).

General Procedure for the 3′ End Modification of Oligo Nucleotides:

The oligos 5-/5Phos/rGrArAr ArArA rArArAr ArA-3 (SEQ ID NO: 1)(oligonucleotide 1) and 5-/5Phos/rGrU/iFluorT/rUrCrG rCrCrArUrU/i6-TAMN/rArArA rArArA rArArA rA-3 (SEQ ID NO: 2) (oligonucleotide2) were purchased from IDT.

-   Procedure A (condensation reactions): A solution containing 10 μM    oligonucleotide in PBS buffer was treated with 50× NaIO₄ (5 mM stock    solution, 500 μM final conc.) and kept at 0° C. for 1 h. The    solution obtained was treated with 100× Na₂SO₃ (10 mM stock    solution, 1 mM final conc.) and brought to RT. After 10 min, 100× of    the nucleophile in water was added (10 mM stock solution, 1 mM final    conc.) and the solution was kept at RT over night. The solution was    desalted using Princeton Separation CS100 spin columns.-   Procedure B (reductive amination reactions): A solution containing    10 μM oligonucleotide in PBS buffer was treated with 50× NaIO₄ (5 mM    stock solution, 500 μM final conc.) and kept at 0° C. for 1 h. The    solution obtained was treated with 200× Na₂SO₃ (10 mM stock    solution, 2 mM final conc.) and brought to RT. After 10 min, 100× of    the nucleophile (10 mM stock solution in water, 1 mM final conc.)    and NaBH₃CN (100 mM stock solution in water, 10 mM final conc.) were    added. The solution was shaken at 37° C. over night and desalted    using Princeton Separation CS100 spin columns.

Analytical data for 3′-modified oligonucleotides are presented in Table7. The following examples were prepared according to the generalprocedure described above.

TABLE 7 Analytical data for 3′-modified oligonucleotides.

Syn- Oligo thetic nucleo- meth- Calc. Exp. No. R1^(a) tide od R_(t) ^(c)mass Mass  1

1 A^(b) 1.92′ 3572.5 [M − H]⁻ 3571.3 [M − H]⁻  2

1 A 1.54′ 3600.4 [M − H]⁻ 3660.2 [M − H]⁻  3

2 A 3.17′ 8180.5 [M − H₃O]⁺ 8180.5 [M − H₃O]⁺  4

1 B 1.83′ 3587.4 [M − H]⁺ 3587.0 [M − H]⁻  5

1 A 1.69′ 3628.4 [M − H]⁺ 3630.1 [M − H]⁻  6

1 B 1.76′ 3614.4 [M − H]⁺ 3613.1 [M − H]⁻  7

1 A 1.32′ 3601.3 [M − H]⁺ 3601.1 [M − H]⁻  8

1 B 2.02′ 3587.3 [M − H]⁻ 3587.4 [M − H]⁻  9

2 A 3.76′ 9040.4 [M − H]⁻ 9037.5 [M − H]⁻ 10

1 A 4.09′ 4926.9 [M − H]⁻ 4928.1 [M − H]⁻ 11

1 B 1.68′ 3806.5 [M − H]⁻ 3806.5 [M − H]⁻ 12

1 A 1.91′ 3699.4 [M − H₃O]⁻ 3699.3 [M − H₃O]⁻ ^(a)Structure shownrepresent one of the possible regioisomeric condensation products.^(b)Compound obtained if no nucleophile was added. ^(c)LCMS method 9

General Procedure for the 3′ End Modification of mRNA:

mRNA in water (0.5-1.5 mg/mL, 90 μL) was treated with NaIIO₄ (0.1 M in 3M NaOAc buffer, pH 5.2, final conc. 10 mM), and the solution was kept onice in the dark for 1 h. The sample was desalted using PrincetonSeparations Centrispin columns, equilibrated with PBS buffer. Thesolution was treated with the appropriate nucleophile (in H₂O or DMSOfor a final concentration of 5 mM), diluted 0.75× with H₂O and shaken at500 rpm at room temperature for 2 h. The resulting solution was desaltedtwice with Princeton Separations CentriSpin 10 columns (PBSequilibrated, then H₂O equilibrated). The RNA solutions obtained werefurther purified by LiCl precipitation.

Biological Data

Analysis of Cap Analog Binding to Biotinylated and Surface ImmobilizedhEIF4E:

Dual histidine and Avi tagged human EIF4E (His6-3C-avi-eIF4E) wasexpressed in 8 L of TB Media. Induction by 0.4 mM IPTG occurred at 2.0OD600, and cells were harvested at 15 OD600. 225 gram pellet was dilutedin buffer (50 mM Tris, 500 mM NaCl, 2 mM MgCl₂, 1 mM TCEP, pH 7.5containing 10% glycerol, protease inhibitors, and DNase) to volume of600 mL and passed once over the microfluidizer. Sample was run on 5 mLHisTrap HP IMAC at 4.0 mL/min IMAC with a 25 mM to 500 mM imidazolegradient for one column volume. GST-PreScission Protease (2 mg, madein-house) was added to sample and allowed to react overnight at 4° C.The cleaved pool was passed over 0.5 mL GST and 0.5 IMAC resin in agravity column. Sample volume was increased to 800 mL using 50 mM TrispH 7.5, 1 mM TCEP and passed over a 5 mL hiTrap SP FF at 4.0 mL/min witha 0 to 1 M NaCl gradient over 20 column volumes. Sample was theninjected onto a 124 mL S75 Gel Filtration Column at 20 mg/mL. FinalAvi-eIF4E (26931 Da) was diluted to 1 mg/mL in 1× Bicine buffer to avolume of 2.5 mg and mixed with ATP/Biotin Mix (10 mM ATP, 10 mMMg(OAc)₂, 50 μM d-biotin final). Biotin Ligase (25 μg BirA produced inhouse) was added to reaction. Reactions were performed with mixing (500rpm) on Eppendorf ThermoMixer R at 30° C. for 60 minutes and checked forcompleteness using LC-MS. To the sample, 100 μl of immobilizedglutathione (1:1 with buffer) was add and mixed for 15 min at 4° C. tobind C3 and Bir3 and removed by centrifugation. The sample was bufferexchanged using two consecutive PD-10 columns equilibrated with 20 mMHEPES, 100 mM KCl, 1 mM DTT, pH 7.5.

Due to low eIF4E stability, the streptavidin coated chip was preparedand run at 10° C. on the Biacore T200. The eIF4E (0.04 mg/mL, 150 μl)was bound to the sample channel of Series S Sensor Chip SA (GE LifeSciences, BR-1005-31) to surface density of 5000 to 7000 RU (ResponseUnits). Buffer flowed over the chip at 30 μL/min, using 1× PBS, 50 mMNaCl, 0.1% Glycerol, 0.1% CHAPS, and 1% DMSO. Samples of various capanalogs were diluted to various concentrations in a range of 100 μM toless than 1 nM. Samples were injected into the Biacore chip with a twominute association time and a five minute dissociation time. Severalbuffer injections were done for each sample for blank subtraction.

Analysis was done for all sets using Biacore T200 evaluation software.Binding analysis for all compounds is reported as response units at 1micromolar compound where a higher value for RU is interpreted asgreater ligand binding to the surface immobilized eIF4E protein. Asubset of compounds was further characterized to determine dissociationconstants using either or both kinetic binding and thermodynamic (SteadyState Affinity) binding analysis. Steady State Affinity fits were donewith default settings (4 seconds before injection stop with 5 secondwindow). Kinetic fits were normally done with 1:1 binding model, withconstant RI=0 and all other variables set to fit globally.

Biacore binding data of cap analogs using the above described method ispresented in Table 8.

TABLE 8 Biacore binding data of cap analogs.

Binding Molecular Level K_(D) K_(D) (μM) Weight (RU) (μM) Steady State/No. —Y—R¹ R⁴ (Da) at 1 μM Kinetic Thermodynamic 1

OH 530.5 127.5 0.1 0.3 2

OH 682.78 14.8 N/D N/D 3

OH 645.82 5.4 N/D N/D 4

OH 612.42 18.0 N/D N/D 5

OH 650.42 3.1 N/D N/D 6

OH 650.42 1.2 N/D N/D 7

OH 649.42 4.9 N/D N/D 8

OH 575.42 12.3 N/D N/D 9

OH 653.32 42.9 N/D N/D 10

OH 559.32 6.6 N/D N/D 11

OH 625.42 11.5 N/D N/D 12

OH 622.42 1.6 N/D N/D 13

OH 612.42 3.8 N/D N/D 14

OH 626.42 1.6 N/D N/D 15

OH 626.42 17.4 N/D N/D 16

OH 616.42 15.4 N/D N/D 17

OH 624.42 16.8 0.03 — 18

OMe 554.89 109.9 0.18 0.16 19

OH 636.42 7.1 N/D N/D 20

OH 624.52 4.3 N/D N/D 21

OH 637.22 2.7 N/D N/D 22

OH 626.42 72.6 N/D N/D 23

OH 613.42 8.6 N/D N/D 24

OH 586.32 37.4 N/D N/D 25

OH 593.42 47.4 N/D N/D 26

OH 598.42 6.7 0.05  4.4 × 10⁻³ 27

OH 674.52 17.3 N/D N/D 28

OH 678.42 56.3 N/D N/D 29

OH 652.42 −2.6 0.03 3.74 × 10⁻³ 30

OH 628.42 10.7 N/D N/D 31

OH 614.52 10.4 N/D N/D 32

OH 722.52 1.0 93.6 0.03 33

OH 660.52 73.9 0.17 0.22 34

OH 669.52 8.1 N/D N/D 35

OH 610.42 13.1 N/D N/D 36

OH 780.62 2.2 N/D N/D 37

OH 594.42 31.5 N/D N/D 38

OH 672.52 29.6 N/D N/D 39

OH 652.52 38.8 N/D N/D 40

OH 751.52 40.6 2.36 1.22 × 10⁻³ 41

OH 757.42 2.0 8.82 0.02 42

OH 751.52 82.5 4.9 1.98 × 10⁻³ 43

OH 674.52 36.6 3.17 0.58 44

OH 646.42 1.5 N/D N/D 45

OH 596.42 9.4 N/D N/D 46

OH 622.42 −3.4 N/D N/D 47

OH 635.42 15.7 N/D N/D 48

OH 622.42 12.7 N/D N/D 49

OH 544.50 53.3 0.02 2.11 × 10⁻³ 50

OH 572.50 3.8 N/D N/D 51

OH 651.52 8.9 N/D N/D 52

OH 661.69 7.3 N/D N/D 53

OH 560.50 149.9 0.19 2.16 × 10⁻² 54

OH 548.40 22.2 2.78 4.75 × 10⁻² 55

OH 666.09 135.7 0.04 0.11 56

OH 648.59 106.8 N/D N/D 57

OH 650.52 83.9 0.03 7.24 × 10⁻³ 58

OH 662.42 95.9 1.00 0.41 59

OH 635.42 7.6 N/D N/D N/D = not determined

FRET Assay for Competitive Binding of Cap Analogs to EIF4E

Materials: His tagged eIF4E protein was purchased from FitzgeraldIndustries International (80R-125; Acton, Mass.) and tRNA was purchasedfrom SIGMA (R5636; St. Louis, Mo.). A biotinylated 20 nucleotide RNAoligo (5′Phos/rGrGrA rCrCrC rCrUrC rUrCrC rCrUrC rCrCrC rCrC biotin-3′)(SEQ ID NO: 3) was purchased from Integrated DNA Technologies(Coralville, Iowa). Europium labeled anti his antibody was purchasedfrom Perkin Elmer (AD0110; Waltham, Mass.) and streptavidin conjugatedalexa fluor 647 was purchased from Life Technologies (S32357; GrandIsland, N.Y.).

TR-FRET Assay for eIF4E Binding:

The TR-FRET assay was performed in an assay buffer of PBS (pH 7.4),0.02% Tween, and 0.1% BSA using four separate addition steps. Initially,5 μl of His-eIF4E (10 nM) and tRNA (0.1 mg/ml) were added into a blackwalled 384 well small volume plate. Next, 1 μl/well of freshly dilutedcompound was added using the Beckman Coulter Biomek FX liquid dispenserwith a final DMSO concentration of 1%. The compound and eIF4E wereallowed to bind for 15 minutes at room temperature. The third additionconsisted of adding 4 μl of chemically capped 7mGDP imidazolemRNA-biotin (10 nM) into every well, with the exception of one columncontaining only buffer to use as a background control. The bindingreaction was incubated for one hour at room temperature. Finally, 5 μlof the europium-labeled anti-His antibody (10 nM) and streptavidinconjugated alexa fluor 647 (10 nM) TR-FRET detection reagents were addedto each well and allowed to incubate at room temperature protected fromlight for 1 h. The plates were read on the Envision plate reader(Perkin-Elmer) to measure signals from both the Alexa fluor (665 nm) andthe europium (615 nm). A ratio of the alexa fluor and europiumfluorescence as a function of cap analog concentration was calculatedfor data analysis and data were fit to a standard EC50 inhibition curvein Graph Pad.

FRET assay data for N⁷-alkylated GMP derivatives using the abovedescribed method is presented in Table 9.

TABLE 9 FRET assay data for N⁷-alkylated GMP derivatives.

No. —Y—R¹ R³ R⁴ AC50/μM* 1

H OH     50.1 2

H OH      8.32 3

H OH     65.1 4

H OH     17.4 5

H OH     87.9 6

Me OH      0.56 7

H OMe      0.85 8

H OH        0.61^(c) 9

H OH — 10

H OH     12.2 11

H OH     14.0 12

H OH     17.8 13

H OH     10.6 14

H OH      5.23 15

H OH     59.5 16

H OH     22.4 17

H OH     42.9 18

H OH     27.7 19

H OH     95.7 20

H OH — 21

H OH >100 22

H OH >100 23

H OH >100 24

H OH >100 25

H OH >100 26

H OH >100 27

H OH >100 28

H OH >100 29

H OH >100 30

H OH >100 31

H OH >100 32

H OH >100 33

H OH >100 34

H OH >100 35

H OH >100 36

H OH >100 37

H OH >100 38

H OH >100 39

H OH >100 40

H OH >100 41

H OH >100 42

H OH >100 43

H OH >100 44

H OH        1.58^(a) 45

H OH      2.59 46

H OH       1.61^(b) 47

H OH       0.61^(b) 48

H OH    100^(d) 49

H OH     39.6 50

H OH      2.11 51

H OH     35.7 52

H OH      2.75 53

H OH     24.6 54

H OH >100 55

H OH     13.8 56

H OH >100 57

H OH >100 58

H OH >100 59

H OH >100 60

H OH >100 61

H OH     16.7 62

H OH >100 63

H OH  >100^(b) 64

H OH >100 65

H H     29.0 66

H OH      5.91 67

H OH      2.69 68

H OH     52.9 69

H OH     63.2 70

H OH     23.7 71

H OH >100 72

H OH >100 73

H OH >100 74

H OH >100 75 Me H OH    25.8^(b) 76

H OH >100 77

H OH >100 78

H OH >100 *values for single measurement are reported unless otherwisenoted. ^(a)average value over 22 measurements ^(b)average value over 2measurements ^(c)average value over 33 measurements ^(d)average valueover 3 measurements

TABLE 10 FRET assay data for N⁷-alkylated GDP derivatives.

AC50/ No. —Y—R¹ R³ R⁴ μM* 1 Me H Me — 2

H H 0.07 3

H H 4.59 4

H H 2.37 5

H H 11.2  6

H H 4.95 7

H H 36.2  8

H H 0.06 9

H H   0.55^(a) 10

H H 1.01 11

H H 20.7  12

H H 14.6  13

H H 27.4  14

H H 75.0  15

H H 0.16 16

H OH 47.1^(a  )  *values for single measurment are reported unlessotherwise noted. ^(a)average value over 2 measurements

TABLE 11 FRET assay data for N⁷-alkylated inosine derivatives.

No. n AC50/μM* 1 1 1.21 2 2   0.49^(a) *values for single measurementare reported unless otherwise noted. ^(a)average value over 2measurements

TABLE 12 FRET assay data for C⁸-substituted purine derivatives. A

B

AC50/ No. Scaffold R⁹ Z₂ μM* 1 A H

33.6- 2 A Me

>100 3 B —

2.1 4 B —

0.3 5 B —

0.70 6 B —

>100 7 B —

>100 8 B —

18.2

Generation of 5′-Monophosphate mRNA:

Standard T7 transcription reactions result in 5′-triphosphate mRNA,which is not compatible with an imidazole phosphate activated cap analogif a triphosphate structure is desired in the final product. In order togenerate a standard triphosphate cap structure using the imidazoleactivated Cap analogs described herein, the RNA substrate must haveeither a 5′-monophosphate mRNA (when using an imidazole-diphosphate capanalog) or 5′-diphosphate mRNA (when using an imidazole-monophosphatecap analog) for chemical capping. In an attempt to generate mono ordiphosphate 5′-terminal mRNA, addition of either 10 mM GMP or 10 mM GDPto the transcription reaction was tested. While 10 mM GMP yielded >95%5′-monophosphate mRNA, 10 mM GDP inhibited the transcription reactionsaltogether. Therefore for generation of 5′-triphosphate Cap mRNAstructures we have focused on the use of 10 mM GMP containingtranscription reactions and imidazole-diphosphate activated cap analogs.

In Vitro Transcription of mRNA (Leptin and Luciferase):

To generate the DNA template for in vitro transcription, the plasmidpGEM-oT7-TEV-oK-Gluc(NcoI)-2hBG-(NotI)-120A orpGEM-oT7-TEV-hLeptin-GAopt-2hBG-120A was linearized with restrictionenzyme BspQ1 (New England Biolabs, Ipswich, Mass.) according tomanufacturer's protocol. The linearized DNA vector was purified byprecipitation with three volumes of 100% ethanol and 1/10 volume of 3MNaOAc, pH 5.1. This was followed by a wash with 70% ethanol and DNA wasresuspended in water.

In vitro transcription was carried out in 40 mM Tris-HCl, pH 8, 8 mMMgCl₂, 1 mM each NTP, 10 mM DTT, 2 mM spermidine, 0.004 U/uL inorganicpyrophosphatase (New England Biolabs, Ipswich, Mass.), 1 U/uL RNaseinhibitor (New England Biolabs, Ipswich, Mass.), 5 U/μL T7 RNApolymerase (New England Biolabs, Ipswich, Mass.), and 0.2 μg/μL oflinearized plasmid DNA.

If mRNA preparation was for 5′ end chemical capping, 10 mM of GMP(Sigma-Aldrich, St. Louis, Mo.) was spiked in the transcriptionreaction. The reaction was incubated at 37° C. for 1.5 h. The DNAtemplate was digested by adding 0.04 U/μL of the TURBO DNase (ThermoFisher, Waltham, Mass.) and incubated at 37° C. for another 30 min. Thetranscript was precipitated by LiCl (final concentration 2.81 mM),followed by a 70% ethanol wash. The pellet was resuspended innuclease-free water.

Chemical Capping of RNA:

RNAs used were either a 20 nucleotide synthetic RNA molecule that wassynthesized with a 5′-monophosphate (Integrated DNA Technologies:5′-P-rGrGrArCrCrCrCrUrCrUrCrCrCrUrCrCrCrCrCrC-3′ (SEQ ID NO: 4)) or5′-monophosphate containing mRNA in vitro transcribed as described above(Gaussia Luciferase mRNA or human leptin mRNA). Both 3′-modified andunmodified RNA was used.

RNA solution was first denatured in water by heating at 65° C. for 10min. The mixture was cooled on ice for 5 min before being added to thecapping buffer (100 mM MES buffer, pH 6.0, 100 mM NaCl and 5 mM MnCl₂).The imidazole activated Cap analog was then added to the reaction to afinal concentration of 5 mM. The reaction was performed at roomtemperature overnight in a Thermoshaker (Grant Instrument, Cambridge,UK). The reaction was quenched by adding EDTA to a final concentrationof 50 mM. The capped product was desalted using Amicon Ultra CentrifugalFilter Unit (Millipore, Billerica, Mass.). The product was furtherpurified with LiCl precipitation (final 2.81 mM LiCl) and washed with70% ethanol. The pellet was resuspended in nuclease-free water.

In some cases chemically capped mRNA was further purified using reversephase HPLC using a Waters XBridge Shield RP18 3.5 um 2.1×100 mm column(Table 13). Mobile Phase A was 0.1 M triethyl ammonium acetate (TEAA) inwater and Mobile Phase B was 0.1 M TEAA in 75% water/25% acetonitrile.The column flow rate was 0.8 mls/minute and the column temperature was65° C. Fractions were collected manually.

TABLE 13 HPLC Purification Gradient. Time (min) % Mobile Phase B 0.1 443.0 44 13 64 14 90 15 90 15.1 44 20 44

LC/MS Analysis of Capping of the 20 Nucleotide Synthetic RNA Oligomer:

Capped and uncapped oligonucleotides were resolved on a Waters AcquityUPLC BEH C18, 1.7 μm, 100 Å, 2.1×75 mm column. The aqueous mobile phasecontained 0.8 μM EDTA, 7.15 mM triethylamine and 192.3 mMhexafluoroisopropanol. The organic mobile phase is methanol. The columnwas kept at 65° C. with flow rate of 0.35 mL/min. A typical gradientramped from 5% to 16% organic in 2 minutes followed by a ramp from16% to25% in 20 minutes. Post HPLC MS analysis of oligonucleotides wasperformed using either the Thermo LTQ-Orbitrap XL or ABSciex 6500 QTrap. The mass spectrometers were run in ESI-MS negative mode scanningfrom 735 to 1550 m/z.

TABLE 14 Efficiency of chemical capping as determined by LC/MS.

% capped with 20 nt oligonucleotide No. —Y—R¹ RNA 1

73 2

91 3

95 4

84 5

>99 6 Me >99 7

87 8

89 9

97 10

98

TABLE 15 Efficiency of chemical capping as determined by LC/MS.

% capped with 20 nt oligonucleotide No. —Y—R¹ RNA 11

65 12 Bn 84

TABLE 16 Efficiency of chemical capping as determined by LC/MS.

% capped with 20 nt No. —Y—R¹ oligonucleotide RNA 13

99

TABLE 17 Efficiency of chemical capping as determined by LC/MS.

% capped with 20 nt oligonucleotide No. —Y—R¹ X RNA 14

CH₂ 87 15

NH 21Transfection of Luciferase mRNA and Luminescence Readout

Cell culture: HEK293 are seeded at a density of 30,000 cells/well in96-well polyD lysine coated plates and incubated at 37° C., 5% CO₂incubator overnight. Culture medium is EMEM (ATCC, cat #30-2003), 10%FBS (Invitrogen), no antibiotic.

mRNA transfection: 100 ng/well of mRNA is transfected using 0.4 μl/wellof DharmaFECT formulation2 (ThermoScientific T-2002-01). This is a 1:4ratio of mRNA to transfection reagent. The mRNA and transfection reagentare mixed in OptiMEM to obtain a final volume of 10 μl/well. The mixtureis incubated at room temperature for 20 minutes. The overnight culturemedium is removed from cells by flipping the plate. 90 μl of freshculture medium is added to each well. 10 μl of mRNA mixture is added toeach well. The plate is incubated at 37° C., 5% CO₂ for 24 hours.

Media collection: Gaussia luciferase protein is secreted in the media.To collect media, 90 μl of media is transferred from the cells into av-bottom 96 well plate. The plate is centrifuged at 1000 rpm for 5minutes. 80 μl of supernatant is transferred into a new v-bottom plateand either frozen or used directly in Luciferase expression assay.

Luciferase expression assay: BioLux Gaussia Luciferase Assay Kit (NewEngland Biolabs Cat #E3300L) is used to perform the Luciferase assay. A1:100 dilution of the BioLux gLUC substrate is made in Assay Buffer (10μl substrate: 1000 μl Assay Buffer). 20 μl of the transfection media isadded to a 96-well white clear bottom plate (Greiner bio-one 655095).The plate is read using the FlexStation 3 Microplate reader (MolecularDevices). At time 0, 50 uL of substrate mix is added to the media plateand data is collected for 60 seconds. The Relative Luminescence Unit(RLU) peaks at 30 seconds and the results are calculated using this timepoint. % luciferase activity is calculated by normalizing the RLU foreach modified mRNA to the RLU for Enzymatically capped Cap-0 mRNA andmultiplying by 100.

Luciferase activity of enzymatically capped HPLC purified mRNA (Cap-1)compared to HPLC purified chemically capped mRNAs Cap-1, Cap-2, andCap-3 is shown in FIG. 1 .

Luciferase activity of enzymatically capped mRNA (Cap-0) compared tochemically capped mRNAs Cap-1, Cap-2, and Cap-3 is shown in FIG. 2 .

Transfection of Capped Leptin mRNA and Luminescence Readout

Cell Culture: HEK293 cells are seeded at a density of 30,000 cells/wellin 96-well polyD lysine coated plates and incubated at 37° C., 5% CO₂incubator overnight. Culture medium is EMEM (ATCC, cat #30-2003), 10%FBS (Invitrogen), no antibiotic.

mRNA Transfection: 100 ng/well of mRNA is transfected using 0.4 μl/wellof DharmaFECT formulation2 (ThermoScientific T-2002-01). This is a 1:4ratio of mRNA to transfection reagent. The mRNA and transfection reagentare mixed in OptiMEM to obtain a final volume of 10 μl/well. The mixtureis incubated at room temperature for 20 minutes. The overnight culturemedium is removed from cells by flipping the plate. 90 μl of freshculture medium is added to each well. 10 μl of mRNA mixture is added toeach well. The plate is incubated at 37° C., 5% CO₂ for 24 hours.

Media collection: The protein is secreted in the media. To collectmedia, 90 μl of media is transferred from the cells into a v-bottom 96well plate. The plate is centrifuged at 1000 rpm for 5 minutes. 80 μl ofsupernatant is transferred into a new v-bottom plate.

Human leptin protein ELISA assay: Human leptin in mouse plasma wasmeasured by ELISA. Antibodies purchased from the R&D systems duoset (Cat#DY398E, part #840279 for capture antibody and part #840280 fordetection antibody) were reconstituted using PBS and titered, againusing PBS. The capture antibody was coated at 4 μg/mL in 30 μl/well on awhite Nunc® Maxisorp 384 well plate (Cat #460372). After an overnightincubation at room temperature the capture antibody was aspirated andthe plate blocked for 2 hours at room temperature with 90 μL/well of KPLmilk blocker (Cat #50-82-00). Once the incubation was completed theplate was aspirated and recombinant standards and samples were added tothe plate at 30 μL/well for 2 hours at 37° C. while shaking at 600 rpm.Sample/standard dilutions were made using casein sample diluent (0.7%Casein, 1.7 mM Sodium Phosphate Monobasic, 8.1 mM sodium phosphatedibasic heptahydrate, 0.15 M NaCl, 0.7% Triton X-100, and 0.1% sodiumazide). Washing/aspiration 3 times with 100 μl/well followed, usingTeknova plate wash solution (Cat #P1192). Next, detection antibody wasdiluted using casein detection antibody diluent (0.4% Casein, 1.7 mMsodium phosphate monobasic, 8.1 mM sodium phosphate dibasicheptahydrate, 0.15 M NaCl, and 0.1% sodium azide) to 12.5 ng/mL andadded at 30 μl/well for 2 hours room temperature. After this incubation,the plate was washed again and a solution of poly-streptavidin-HRP (Cat#21140) at a 1:1250 dilution in HRP dilution buffer (1.7 mM sodiumphosphate monobasic, 8.1 mM sodium phosphate dibasic heptahydrate, 0.145M NaCl, 0.1% chloroacetamide, 1% BSA Protease Free, and 0.05% Tween 20)was added to each well (30 μL/well) and incubated for 30 minutes roomtemperature. A final wash/aspiration removed the HRP solution and achemiluminescent substrate was added at 30 μL/well (Cat #1859678 &1859679). The plate was quickly read using a SpectramaxM5 plate readerwith a 50 ms integration time. The dynamic range of the ELISA is from5-150 pM of human leptin.

Leptin expression data with chemically capped HPLC Purified mRNAs Cap-1and Cap-2 is shown in FIG. 3 .

HEK293 Cell S100 Extract: S100 extract was prepared following standardprotocols for S100 preparation. HEK293 (Thermo Scientific SH30521.02)cells were grown in 80% FreeStyle 293 (Invitrogen 12338) and 20% SFM4 in8% CO₂ with humidified atmosphere on an orbit shaker rotating at 100rpm. 2.5×10⁹ cells were pelleted at 1,500 rpm for 5 minutes in aclinical centrifuge. Pellet was washed in 1 L ice-cold DPBS and spunagain at 1,500 rpm for 10 minutes at 4° C. in the clinical centrifuge.Supernatant was removed and cell pellet was resuspended in 250 ml ofice-cold PBS, broken up by pipetting and spun at 1,700 rpm for 6 minutesat 4° C. in the clinical centrifuge. Supernatant was removed and thepellet was resuspended in 5 volumes of Buffer A (10 mM HEPES-KOH, pH7.9, 1.5 mM MgCl₂, 10 mM KCl, freshly added 0.5 mM DTT). Cells wereincubated on ice for 10 minutes, spun at 2,000 rpm for 10 minutes andsupernatant was removed. An additional volume of Buffer A was added andthe pellet was broken up using 5 strokes of a loose pestle douncehomogenizer. Cells were lysed with 15 strokes of a tight pestle douncehomogenizer and lysis was confirmed using a microscope. Lysed materialwas spun at 4,500 rpm for 10 min at 4° C. in the clinical centrifuge.The supernatant was removed and used to make S100 extract. 0.11 volumeof Buffer B (0.3 M HEPES-KOH, pH 7.9, 1.4 M KCl, 30 mM MgCl₂) was addedto S100, mixed gently by inversion, and spun in ultracentrifuge at102,000 g for 1 hour at 4° C. Supernatant was removed and placed in achilled 15 mL conical tube. Clear supernatant from the bottom layerbelow the lipid layer was removed and dialysed against 1 L Buffer D (20mM HEPES-KOH, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT,0.5 mM PMSF) for >5 hours at 4° C. S100 extract was stored at 80° C. insmall aliquots until ready for use.

FRET RNA degradation assay: The RNA degradation assay was performedfollowing the protocol in Uhler et al J. Am. Chem. Soc. 2003, 125,14230-14231 with some modifications. The oligos5-/5Phos/rGrU/iFluorT/rUrCrG rCrCrA rUrU/i6-TAMN/rArArA rArArA rArArArA-3 (SEQ ID NO: 5) and 5-/5Phos/rGrU/iFluorT/rUrCrG rCrCrArUrU/i6-TAMN/rArArA rArArA rArArA rA/3BIO-3 (SEQ ID NO: 6) werepurchased from IDT and used in the RNA degradation assay. The assaybuffer consisted of 130 mM K-glutamate pH 7.5, 1 mM MgCl₂. DTT was addedto a final concentration of 10 mM just prior to experiment. 100 uM or200 uM oligo was used depending on the reaction volume 25 uL and 50 uL,respectively. S100 extract varied from 0.3%-20% v/v and Buffer D was 20%of total reaction volume for all samples. All reactions were run in 384well plate at 37° C. At 100 sec, S100 extract was added to the oligobuffer mix. Fluorescein was excited at 490 nm and fluorescence emissionwas measured at 520 nm and 585 nm. Data were collected every minute for2 hours. The FRET ratio Q (=F₅₈₅/F₅₂₀) was calculated and normalized tothe initial value Q₀. The data were fit to the single-exponential decayfunction y=y_(o)−Plateau*exp(−K*X)+Plateau using Prism 5.0. To extractMichaelis-Menten parameters, the dependence of the rate constant k_(dec)on % S100 [X] was fit to the Michaelis-Menten equation Y=V max*X/(Km+X)using Prism 5.0.

TABLE 18 Stability of FRET RNA oligo in 1.2% HEK293 S-100 extract.

3′ end No. Z₃ compound T_(1/2) (s) 1

Un- modified 424 ± 65  2

biotin 1046 ± 176  3

Exp10 3563 ± 1486 4

Exp11 1906 ± 648  5

Exp25 879 6

Exp30 1006 7

Exp39 2464 8

Exp40 275940 9

Exp41 23160

3′ End Modification of mRNA

Transfection of Luciferase 3′ End Modified mRNA and LuminescenceReadout:

Cell culture: HEK293 cells are seeded at a density of 30,000 cells/wellin 96-well polyD lysine coated plates and incubated at 37 C, 5% CO₂incubator overnight. Culture medium is EMEM (ATCC, cat #30-2003), 10%FBS (Invitrogen), no antibiotic.

mRNA transfection: 30 ng/well of mRNA is transfected using 0.4 μl/wellof DharmaFECT formulation2 (ThermoScientific T-2002-01). The mRNA andtransfection reagent are mixed in OptiMEM to obtain a final volume of 10μl/well. The mixture was incubated at room temperature for 20 minutes.The overnight culture medium was removed from cells by flipping theplate. 90 μl of fresh culture medium was added to each well and 10 μl ofmRNA mixture was added to each well. The plate is incubated at 37° C.,5% CO₂ for 24 hours.

Media collection: Gaussia luciferase protein is secreted in the media.To collect media, 24 hours after transfection, 90 μl of media wastransferred from the cells into a v-bottom 96 well plate and centrifugedat 1000 rpm for 5 minutes. 80 μl of supernatant was transferred into anew v-bottom plate and either frozen or used directly in Luciferaseexpression assay or leptin elisa.

Luciferase expression assay: BioLux Gaussia Luciferase Assay Kit (NewEngland Biolabs Cat #E3300L) is used to perform the Luciferase assay. A1:100 dilution of the BioLux gLUC substrate is made in Assay Buffer (10μl substrate: 1000 μl Assay Buffer). 20 μl of the transfection media isadded to a 96-well white clear bottom plate (Greiner bio-one 655095).The plate is read using the FlexStation 3 Microplate reader (MolecularDevices). At time 0, 50 uL of substrate mix is added to the media plateand data is collected for 60 seconds. The Relative Luminescence Unit(RLU) peaks at 30 seconds and the results are calculated using this timepoint. % luciferase activity is calculated by normalizing the RLU foreach modified mRNA to the RLU for enzymatically capped Cap1 mRNA andmultiplying by 100.

TABLE 19 3′ modified GLuc-mRNA expression data in HEK293 cells.

% 3′ end Luciferase No. Z₃ compound activity* 1

Unmodified 100 2

biotin 443.5 ± 97   4

Exp048 311 5

Exp051 451 ± 136 6

Exp049 287 7

Exp050 513 ± 341 8

Exp057 268 9

Exp058 268 *normalized to umodified 24 hours after transfection.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A compound of Formula I or a pharmaceutically acceptable salt thereof

wherein Z₁ is:

Z₂ is absent or a linking moiety selected from the group consisting of —O—, —S—, lower alkyl, aminoalkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocyclyl,

A₁ and A₃ are independently selected from the group consisting of absent, NH, S, and O; A₂ is absent or selected from the group consisting of >CR⁶R⁷, >NR⁶, >NNR⁶R⁷, >NOR⁶, >S, and >O; Y is absent or a linking moiety selected from the group consisting of lower alkyl, alkenyl, alkynyl, —(CH₂)_(n)OR¹⁵, —(CH₂)_(n)COOR¹⁵, and —(CH₂)_(n)C(O)NR¹²; R¹ is selected from the group consisting of H, substituted cycloalkyl, substituted cycloalkenyl, substituted aryl, substituted heteroaryl, and substituted heterocyclyl; R⁵, R⁶, and R⁸ are independently selected from the group consisting of H, substituted alkyl, polyamine, PEGs, —(CH₂)_(n1)NR¹²R¹³, —(CH₂)_(n1)NR¹⁴C(O)R¹⁵, —(CH₂)_(n1)OR¹⁵, —(CH₂)_(n1)C(O)OR¹⁵, —(CH₂)_(n1)C(O)R¹⁵, —(CH₂)_(n1)C(O)NR¹²R¹³, —O—(CH₂)_(n3)—C(O)—(NR¹²)₂—C(O)—X₂, —O—(CH₂)_(n3)—C(O)—[NR¹²—C(O)—(CH₂)_(n3)]₁₋₃—X₂, or R⁶ and R⁸ together form a ring that is substituted and contains 10-80 ring atoms in which 10-40 ring atoms can be hetero atoms, or R⁶ and R⁷ together form a 3-8 membered ring that is substituted and in which 1 to 6 ring atoms can be hetero atoms; R⁷, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently selected from the group consisting of H, substituted lower alkyl, and substituted acyl; R⁹ is selected from the group consisting of H and substituted lower alkyl; R¹⁰ is selected from the group consisting of H, —NR¹²R¹³, and —OR¹⁶; n is 1 to 4; n1 is zero to 10; n2 is 1 to 12; n3 is 1 to 8; X is selected from the group consisting of O, S, NH, and substituted alkanediyl; X₂ is selected from the group consisting of affinity moiety and detection moiety, and X_(n) is a nucleobase.
 2. (canceled)
 3. (canceled)
 4. The compound of claim 1, which has the following formula

wherein A₂ is absent; and the other variables are as defined in Formula I. 5-9. (canceled)
 10. The compound of claim 1, which has the following formula

wherein -A₁-R⁵ and -A₃-R⁸ are —OH; A₂ is absent; X_(n) is a nucleobase, and the other variables are as defined in Formula I. 11-14. (canceled)
 15. A compound of Formula XI or a pharmaceutically acceptable salt thereof

wherein Z₂ is absent or a linking moiety selected from the group consisting of —O—, —S—, lower alkyl, aminoalkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocyclyl,

Y is absent or a linking moiety selected from the group consisting of lower alkyl, alkenyl, alkynyl, —(CH₂)_(n)OR¹⁵, —(CH₂)_(n)COOR¹⁵, and —(CH₂)_(n)C(O)NR¹²; R¹ is selected from the group consisting of H, substituted cycloalkyl, substituted cycloalkenyl, substituted aryl, substituted heteroaryl, and substituted heterocyclyl; R⁹ is selected from the group consisting of H and substituted lower alkyl; R¹⁰ is selected from the group consisting of H, —NR¹²R¹³, and —OR¹⁶; R¹², R¹³, R¹⁵, and R¹⁶ are independently selected from the group consisting of H, substituted lower alkyl, and substituted acyl; X is selected from the group consisting of O, S, NH, and substituted alkanediyl; Z is selected from the group consisting of —OH,

n is 1 to 4; n2 is 1 to 12; and n4 is 0-2.
 16. The compound of claim 15, comprising a structure selected from the group consisting of: 